β-substituted γ-amino acids and analogs as chemotherapeutic agents

ABSTRACT

β-Substituted γ-amino acids, β-substituted γ-amino acid derivatives, and β-substituted γ-amino acid analogs and (bio)isosteres and their use as chemotherapeutic agents are disclosed. The β-substituted γ-amino acid derivatives and β-substituted γ-amino acid analogs and (bio)isosteres are selective LAT1/4F2hc substrates, capable of passing through the blood-brain barrier, and exhibit rapid uptake and retention in tumors expressing the LAT1/4F2hc transporter. Methods of synthesizing the β-substituted γ-amino acid derivatives and β-substituted γ-amino acid analogs and (bio)isosteres and methods of using the compounds for treating tumors are also disclosed. The β-substituted γ-amino acid derivatives and β-substituted γ-amino acid analogs and (bio)isosteres exhibit an improved selectivity toward tumor cells expressing the LAT1/4F2hc transporter and accumulate in cancerous cells when administered to a subject in vivo. The β-substituted γ-amino acid derivatives and β-substituted γ-amino acid analogs and (bio)isosteres exhibit an increased efficacy on a variety of tumor types.

This application is a Continuation of U.S. application Ser. No.15/181,020 filed on Jun. 13, 2016, now allowed, which is a Continuationof U.S. application Ser. No. 14/613,130 filed on Feb. 3, 2015, issued asU.S. Pat. No. 9,394,236, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/935,235, filed on Feb. 3,2014, which is incorporated by reference in its entirety.

FIELD

Disclosed herein are β-substituted γ-amino acid derivatives andβ-substituted γ-amino acid analogs and (bio)isosteres and their use astherapeutic agents. The β-substituted γ-amino acid derivatives andβ-substituted γ-amino acid analogs and (bio)isosteres are selectivesubstrates for LAT1/4F2hc, capable of passing through the blood-brainbarrier, and exhibit rapid uptake and retention in tissue such as tumorsexpressing the LAT1/4F2hc transporter. Pharmaceutical compositionscomprising the β-substituted γ-amino acid derivatives and β-substitutedγ-amino acid analogs and (bio)isosteres and uses thereof are alsodisclosed.

BACKGROUND

The ability to selectively target chemotherapy has immense value inclinical practice. Cancer is a leading cause of death in the developedworld, with one in every three people developing cancer during his orher lifetime. There are many treatment options for cancer includingsurgery, chemotherapy, radiation therapy, immunotherapy, and monoclonalantibody treatment. Unfortunately, for many patients cancer treatmentoptions are limited and response rates remain low.

Surgery is the oldest effective form of tumor therapy and can oftenresult in a complete cure, depending of the type and nature of thetumor. Many tumors, however, occur in locations and/or number that makesurgery impossible or impractical. Also, surgical debulking is notguaranteed to remove all abnormal cells, particularly in the case oftumors located in the brain where maximum preservation of normal tissueis desired. Residual abnormal cells pose an increased risk of tumorre-growth and/or metastasis.

Radiation therapy is often used as an adjunct to surgery. Various typesof radiation, both from external and implanted sources, have been usedwith some success. Low linear-energy-transfer (LET) sources, such asβ-particles and γ-rays, require repeated treatments over extendedperiods of time to produce any significant reduction in tumor cells.High LET sources, such as neutrons, protons or α-particles, do notrequire oxygen to enhance their biological effectiveness. External beamtherapy has been available for decades, however, significant radiationdamage occurs to normal tissues, and patients often succumb towidespread radiation-induced necrosis (Laramore, et al., Cancer, 1978,42(1), 96-103).

Chemotherapy is used in attempts to cure or palliate cancer. Smallmolecule chemotherapeutics target rapidly dividing cells, halting cellproliferation by interfering with DNA replication, cytoskeletalrearrangements and/or signaling pathways that promote cell growth.Disruption of cell division slows the growth of malignant cells and mayalso kill tumor cells by triggering apoptosis. Alkylating agents, suchas bis(2-chloroethyl)amine derivatives, act by covalent interaction withnucleophilic heteroatoms in DNA or proteins. It is believed that thesedifunctional agents are able to crosslink a DNA chain within a doublehelix in an intrastrand or interstrand fashion, or to crosslink betweenDNA, proteins or other vital macromolecules. The crosslinking results ininhibitory effects on DNA replication and transcription with subsequentcell death. Since these drugs also indiscriminately kill normalpopulations of rapidly proliferating cells, such as those found in theimmune system and in the gastrointestinal tract, side effects that limittolerated doses, are common.

The harsh side effects and the ultimate failure of most chemotherapyregimens have motivated investigation of alternatives, including drugsthat target specifically tumor cells. Normal cells and tumor cellsdiffer markedly in nutrient and energy metabolism, a phenomenon known asthe Warburg effect (Ganapathy, et al., Pharmacol Ther, 2009, 121(1),29-40; and Vander Heiden, et al., Science, 2009, 324(5930), 1029-1033).Enhanced proliferation in tumor cells places increased demand fornutrients to serve as building blocks for the biosynthesis ofmacromolecules and as sources of energy. Tumor-selective nutrientaccumulation is most clearly evident in imaging studies of human tumorsusing positron emission tomography (PET) and [¹⁸F]-fluorodeoxyglucose(FDG). FDG accumulates at high levels in many kinds of solid tumors andis thought to be taken up into tumor cells by sugar transporters. Aminoacids are the primary source of cellular nitrogen, used for nucleotide,glutathione, amino sugar, and protein synthesis. In addition, tumorsoften utilize the carbon skeletons of amino acids as an oxidative fuelsource for ATP generation in addition to glucose and fatty acids(Baggetto and Biochimie, 1992, 74(11), 959-974; Mazurek and Eigenbrodt,2003, Anticancer Res, 2003, 23(2A), 1149-1154; and DeBerardinis, et al.,Proc Natl Acad Sci USA, 2007, 104(49), 19345-19350). Therefore, tumorcells must express select specific transporters to satisfy maintenanceand growth requirements for nutritional amino acids. To compete withsurrounding tissue for nutrients, tumor cells up-regulate levels ofcertain transporters to allow for more efficient extraction of nutrientsthan that of the host tissue.

Amino acid transport across the plasma membrane in mammalian cells ismediated by different transport “systems” such as the sodium-dependentsystems A, ASC and N, and sodium-independent system L (Christensen, PhysRev, 1990, 70, 43-77). System L is a ubiquitous plasma membrane aminoacid transport system that is characterized by the sodium-independentuptake of bulky, hydrophobic amino acids and its high affinityinteraction with 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH).System L activity is presently attributed to four sodium-independenttransporters (LAT1-4). However, most cancers over-express only onemember, the large amino acid transporter 1 (LAT1/4F2hc). Thistransporter is a heterodimer consisting of a light chain (LAT1) thatconstitutes the transporter and a heavy chain 4F2hc (also known as CD98,or Tumor Antigene TA1) that is required for proper targeting of thelight chain to the plasma membrane. The expression and activity ofLAT1/4F2hc correlates with cell proliferation and cancer growth; andup-regulation of LAT1/4F2hc has been observed, for example, in cancersof brain, colon, lung, liver, pancreas, and skin (Jager, et al., J NuclMed, 1998, 39(10), 1736-1743; Ohkame, et al., J Surg Oncol, 2001, 78(4),265-267; Tamai, et al., Cancer Detect Prev, 2001, 25(5), 439-445; Kim,et al., Anticancer Res, 2004, 24(3a), 1671-1675; Kobayashi, et al.,Neurosurgery, 2008, 62(2), 493-503; Imai, et al., Histopathology, 2009,54(7), 804-813; and Kaira, et al., 2009, Lung Cancer, 66(1), 120-126).Furthermore, the expression of LAT1/4F2hc has been used as anindependent factor to predict poor prognoses in patients with astrocyticbrain tumors, lung cancer, and prostate cancer (Nawashiro, et al., Int JCanc, 2006, 119(3), 484-492; Kaira, et al., Lung Cancer, 2009, 66(1),120-126; Kaira, et al., Cancer Sci, 2008, 99(12), 2380-2386; and Sakata,et al., Pathol Int, 2009, 59(1), 7-18). Inhibition ofLAT1/4F2hc-mediated transport with non-metabolizable amino acids such asBCH can reduce growth and induce apoptosis in cancer cells in vitro(Kim, et al., Biol Pharm Bull, 2008, 31(6), 1096-1100; Shennan andThomson, Oncol Rep, 2008, 20(4), 885-889; and Kaji, et al., Int JGynecol Cancer, 2010, 20(3), 329-336). Clinical studies have shown thatthe specificity and positive predictive value ofL-[3-¹⁸F]-α-methyltyrosine ([¹⁸F]-FAMT) PET is superior to [¹⁸F]-FDGPET. The uptake of [¹⁸F]-FAMT in tumors has been closely correlated withLAT1 expression (Haase, et al., J Nucl Med, 2007, 48(12), 2063-2071;Kaira, et al., Clin Cancer Res, 2007, 13(21), 6369-6378; and Urakami, etal., Nucl Med Biol, 2009, 36(3), 295-303).

In particular, melphalan is an effective chemotherapy drug used intreating multiple myeloma, ovarian cancer, retinoblastoma, and otherhematopoietic tumors. However, substrates such as gabapentin arereported to be transported much more rapidly than melphalan (Uchino, etal., Mol Pharmacol 2002, 61(4), 729-737). It is widely believed thatuptake of melphalan (Alkeran®, otherwise known as L-PhenylalanineMustard, or L-PAM) into cells is mediated by amino acid transporters.Melphalan is an alkylating agent linked to the essential amino acidphenylalanine. Because normal cells and tumor cells differ markedly innutrient and energy metabolism (Warburg effect) (Vander Heiden, et al.,Science, 2009, 324(5930), 1029-1033), melphalan was introduced intoclinical practice with the expectation that it would preferentiallyaccumulate in rapidly dividing tumor cells compared to normal cells,thereby increasing its overall therapeutic index. Surprisingly,melphalan caused many of the same side effects as other conventionalalkylation agents, including myelosuppression. In a series ofpublications, Vistica et al. examined melphalan transport in differentcell types and identified two independent transport systems formelphalan. One system, presumed to be System L, is characterized by thesodium-independent uptake of bulky, hydrophobic amino acids and itssensitivity toward inhibition with2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) (Vistica, BiochimBiophys Acta, 1979, 550(2), 309-317). A second transport system issodium-dependent, exhibits its highest affinity for leucine, but isinsensitive to both BCH and the system A-specific inhibitorα-amino-isobutyric acid (AlB) (Vistica, Biochim Biophys Acta, 1979,550(2), 309-317). Although LAT1 is overexpressed on the cell surface ofalmost all tumor cells regardless of the tissue of origin, responserates to melphalan are low for most cancer types, and the drug is onlyapproved for the treatment of multiple myeloma and ovarian cancer.Melphalan is a poor substrate for LAT1 compared to other large aminoacids such as phenylalanine and leucine (Uchino, et al., Mol Pharmacol2002, 61(4), 729-737; and Hosoya, et al., Biol Pharm Bull, 2008, 31(11),2126-2130). Nitrogen mustard derivatives with higher selectivity towardthe LAT1/4F2hc system could reduce side effects associated with nitrogenmustard therapy, allow for an increase in dose, and extend the use intoother areas of cancer treatment.

Although the potential for active transport strategies for increasingdrug uptake into tumor cells is known and generally accepted,chemotherapeutics and tumor imaging agents have in general not beenoptimized for transporters known to be over-expressed in tumor cells.While the general concept of using LAT1/2Fhc-selective compounds todeliver therapeutic agents to tumors is appreciated, the existing artgives no guidance as to how one prepares a composition that exploitsLAT1/4F2hc selective compounds. Thus, there is a need for newtherapeutic agents that are more selective toward LAT1/4F2hc.

Several amino acid-related drugs that are substrates of the LAT1/4F2hctransporter are known, including L-Dopa, 3-O-methyldopa, droxidopa,carbidopa, 3,3′,5′-triiodothyronine, thyroxine, gabapentin, andmelphalan (Uchino, et al., Mol Pharm 2002, 61(4), 729-737; and del Amoet al., Eur J Pharm Sci, 2008, 35(3), 161-174).

SUMMARY

Differentiation of malignant cancer tissue from neighboring nonmalignanttissue can be accomplished by exploiting changes in biochemical fluxesthat occur in response to metabolic, genetic, and/or microstructuralchanges in the malignant cells. Compounds provided by the presentdisclosure substantially improve chemotherapy of tissue expressing theLAT1/4F2hc transporter including malignant tumors. The β-substitutedγ-amino acid derivatives, β-substituted γ-amino acid analogs, andβ-substituted γ-amino acid carboxylic acid (bio)isosteres provided bythe present disclosure provide greater uptake selectivity for the targettissue or cells expressing the LAT1/4F2hc transporter with lownon-specific uptake for non-target tissues or cells.

Embodiments provided by the present disclosure provide novelβ-substituted γ-amino acid derivatives and β-substituted γ-amino acidanalogs, and methods of using such derivatives, for example, aschemotherapeutic agents. Certain embodiments further relate to methodsof synthesizing β-substituted γ-amino acid derivatives and β-substitutedγ-amino acid analogs and to pharmaceutical compositions comprising suchderivatives. The β-substituted γ-amino acid derivatives andβ-substituted γ-amino acid analogs provided by the present disclosureexhibit selectivity for LAT1/4F2hc and therefore accumulate in cancerouscells when administered to a subject in vivo. Advantages provided bycompounds of the present disclosure reflect the properties of LAT1/4F2hcsubstrates, namely, blood brain-barrier (BBB) permeability, rapiduptake, and prolonged retention in tumors and further serve aschemotherapeutic agents with improved therapeutic index and safety.

In a first aspect, compounds of Formula (1) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

at least one of R¹ and R⁵ is independently selected from halogen,—N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(OR¹⁰)(R¹⁰), —NO₂, —NO, —N(R¹⁰)(S(═O)R¹⁰),—N(R¹⁰)(S(═O)₂R¹⁰), —N(R¹⁰)(C(O)R¹⁰), —N(R¹⁰)(C(O)OR¹⁰),—N(R¹⁰)(C(O)N(R¹⁰)₂, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, —SH, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, —S(O)N(R¹⁰)₂,—S(O)₂N(R¹⁰)₂, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, C₃₋₆cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyloxy, substitutedC₃₋₆ cycloalkyloxy, C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂cycloalkylalkyl, C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, C₇₋₁₆ arylalkyl,substituted C₇₋₁₆ arylalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ heteroalkoxy, substituted C₁₋₆ heteroalkoxy, C₃₋₆heterocycloalkyl, substituted C₃₋₆ heterocycloalkyl, C₄₋₁₂heterocycloalkylalkyl, substituted C₄₋₁₂ heterocycloalkylalkyl, C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₆₋₁₆ heteroarylalkyl, andsubstituted C₆₋₁₆ heteroarylalkyl;

one of R¹, R², R³, R⁴, and R⁵ comprises a chemotherapeutic moiety;

each of the other of R¹, R², R³, R⁴, and R⁵ is independently selectedfrom hydrogen, deuterio, halogen, —OH, —N(R¹⁰)₂, —NO₂, —NO, —CN,—COOR¹⁰, —CON(R¹⁰)₂, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₆ cycloalkyl,substituted C₃₋₆ cycloalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, C₁₋₆ heteroalkoxy,substituted C₁₋₆ heteroalkoxy, C₄₋₈ cycloalkylalkyl, and C₄₋₈cycloalkylheteroalkyl;

R⁶ is selected from a carboxylic acid (—COOH), a carboxylic acid analog,and a carboxylic acid (bio)isostere;

each R⁷ is independently selected from hydrogen, deuterio, halogen,hydroxyl, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, and phenyl; or two R⁷together with the carbon to which they are bonded form a ring selectedfrom a C₃₋₆ cycloalkyl ring and a C₃₋₆ heterocycloalkyl ring;

R⁸ is selected from hydrogen, deuterio, halogen, C₁₋₆ alkyl, substitutedC₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl, C₁₋₆ alkoxy,substituted C₁₋₆ alkoxy, C₁₋₆ heteroalkoxy, substituted C₁₋₆heteroalkoxy, C₃₋₆ cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆cycloalkyloxy, substituted C₃₋₆ cycloalkyloxy, —OH, —COOR¹⁰, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₆ cycloalkyl, and phenyl;

each R¹⁰ is independently selected from hydrogen, deuterio, C₁₋₄ alkyl,and C₁₋₄ alkoxy, or two geminal R¹⁰ together with the nitrogen to whichthey are bonded form a 3- to 6-membered heterocyclic ring; and

L is —(X)_(a)—, wherein,

-   -   each X is independently selected from a bond (“-”), —C(R¹⁶)₂—,        wherein each R¹⁶ is independently selected from hydrogen,        deuterio, halogen, hydroxyl, C₁₋₄ alkyl, and C₁₋₄ alkoxy, or two        R¹⁶ together with the carbon to which they are bonded form a        C₃₋₆ cycloalkyl ring or a C₃₋₆ heterocycloalkyl ring, —O—, —S—,        —SO—, —SO₂—, —CO—, and —N(R¹⁷)—, wherein R¹⁷ is selected from        hydrogen and C₁₋₄ alkyl; and    -   a is selected from 0, 1, 2, 3, and 4.

In a second aspect, chemotherapeutic moieties are provided, comprising achemotherapeutic moiety of chemotherapeutic drugs known in the art thatretains cytotoxic activity when bonded through a spacing moiety, e.g.,an aryl ring and a linker L, to a γ-amino acid derivative, γ-amino acidanalog, or γ-amino acid carboxylic acid (bio)isostere as a LAT1recognition element provided by the present disclosure. The conjugate orfusion product of the chemotherapeutic moiety with the γ-amino acidderivative, γ-amino acid analog, or γ-amino acid carboxylic acid(bio)isostere is a selective substrate for the LAT1/4F2hc transporter.

In a third aspect, chemotherapeutic moieties are provided wherein thechemotherapeutic moiety is selected from a nitrogen mustard—N(—CR₂—CR₂—X)₂, a N-monoalkyl or N,N-dialkyl triazene (—N═N—NR₂), ahaloacetamide (—NR—CO—CH₂—X), an epoxide (—CROCR—R), an aziridine(—NC₂H₄), a Michael acceptor (—CR═CR-EWG-), a sulfonate or abissulfonate ester (—OSO₂R or ROSO₂—), an N-nitrosourea (—NR—CO—N(NO)R),a bissulfonyl hydrazine (R″SO₂—NR—N(−)—SO₂R′″, —SO₂—NR—NR′—SO₂R′″, orR″SO₂—NR—NR′—SO₂—), a phosphoramidate (—O—P(═O)(N(R)—CH₂—CH₂—X)₂ or—O—P(═O)(N(—CH₂—CH₂—X)₂)₂, and a radionuclide such as, for example,131-iodine (¹³¹[I]—) or 211-astatine (²¹¹[At]-).

In a fourth aspect, chemotherapeutic moieties of Formula (2) areprovided:

wherein,

A is selected from a bond (“-”), oxygen (—O—), sulfur (—S—), amino(—NR¹⁰—), methylene (—CH₂—), methyleneoxy (—CH₂—O—), oxycarbonyl(—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl (—NR¹⁰—C(═O)—),oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl (—S—C(═S)—),aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl (—CH₂—O—C(═O)—),methylenethiocarbonyl (—CH₂—S—C(═O)—), methyleneaminocarbonyl(—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl (—CH₂—O—C(═S)—),methylenethiothiocarbonyl (—CH₂—S—C(═S)—), methyleneaminothiocarbonyl(—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—), methylencarbonyl (—CH₂—C(═O)—),thiocarbonyl (—C(═S)—), and methylenthiocarbonyl (—CH₂—C(═S)—);

Z is selected from a bond (“-”) and oxygen (—O—);

Q is selected from —O⁻ (a negatively charged oxygen atom) that is boundto a positively charged nitrogen atom) and a free electron pair (:),with the proviso that when Q is —O⁻ (a negatively charged oxygen atomthat is bound to a positively charged nitrogen atom), A is selected froma bond (“-”) and methylene (—CH₂—),Z is a bond (“-”), and thechemotherapeutic moiety of Formula (2) is an N-oxide(-A-N⁺(—O⁻)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)₂);

each R¹¹ is independently selected from hydrogen, deuterio, and C₁₋₃alkyl; and

each R⁹ is independently selected from fluoro (—F), chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, whereinR⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl), and (substituted) arylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₆₋₁₀ aryl).

In a fifth aspect, pharmaceutical compositions are provided comprising acompound of Formula (1) and at least one pharmaceutically acceptablevehicle.

In a sixth aspect, methods for treating cancer in a patient comprising,administering a compound of Formula (1) or a pharmaceutical compositioncomprising a compound of Formula (1) to a patient in need of suchtreatment comprising administering a therapeutically effective amount ofthe compound to the patient are provided.

DETAILED DESCRIPTION Definitions

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a moiety or substituent. For example,—CONH₂ is attached through the carbon atom.

“Alkyl” refers to a saturated or unsaturated, branched, orstraight-chain, monovalent hydrocarbon radical derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkene,or alkyne. Examples of alkyl groups include methyl; ethyls such asethanyl, ethenyl, and ethynyl; propyls such as propan-1-yl, propan-2-yl,prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl, 2-methyl-propan-2-yl, but-1-en-1-yl,but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, but-1-yn-1-yl, but-1-yn-3-yl,but-3-yn-1-yl, etc.; and the like. The term “alkyl” is specificallyintended to include groups having any degree or level of saturation,i.e., groups having exclusively carbon-carbon single bonds, groupshaving one or more carbon-carbon double bonds, groups having one or morecarbon-carbon triple bonds, and groups having combinations ofcarbon-carbon single, double, and triple bonds. Where a specific levelof saturation is intended, the terms alkanyl, alkenyl, and alkynyl areused. In certain embodiments, an alkyl group is C₁₋₆ alkyl, C₁₋₅ alkyl,C₁₋₄ alkyl, C₁₋₃ alkyl, and in certain embodiments, ethyl or methyl.

“Alkylsulfanyl” also referred to as “alkylthio”, refers to a radical —SRwhere R is alkyl or cycloalkyl as defined herein. Examples ofalkylsulfanyl groups include methylsulfanyl, ethylsulfanyl,propylsulfanyl, isopropylsulfanyl, butylsulfanyl, andcyclohexylsulfanyl. In certain embodiments, an alkylsulfanyl group isC₁₋₆ alkylsulfanyl, in certain embodiments, C₁₋₅ alkylsulfanyl, incertain embodiments, C₁₋₄ alkylsulfanyl, in certain embodiments, C₁₋₃alkylsulfanyl, in certain embodiments, ethylsulfanyl (ethylthio), and incertain embodiments, methylsulfanyl (methylthio).

“Alkylsulfinyl” refers to a radical —S(O)R where R is alkyl orcycloalkyl as defined herein. Examples of alkylsulfinyl groups includemethylsulfinyl, ethylsulfinyl, propylsulfinyl, isopropylsulfinyl,butylsulfinyl, and cyclohexylsulfinyl. In certain embodiments, analkylsulfinyl group is C₁₋₆ alkylsulfinyl, in certain embodiments, C₁₋₅alkylsulfinyl, in certain embodiments, C₁₋₄ alkylsulfinyl, in certainembodiments, C₁₋₃ alkylsulfinyl, in certain embodiments, ethylsulfinyl,and in certain embodiments, methylsulfinyl.

“Alkylsulfonyl” refers to a radical —S(O)₂R where R is alkyl orcycloalkyl as defined herein. Examples of alkylsulfonyl groups includemethylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl,butylsulfonyl, and cyclohexylsulfonyl. In certain embodiments, analkylsulfonyl group is C₁₋₆ alkylsulfonyl, in certain embodiments, C₁₋₅alkylsulfonyl, in certain embodiments, C₁₋₄ alkylsulfonyl, in certainembodiments, C₁₋₃ alkylsulfonyl, in certain embodiments, ethylsulfonyl,and in certain embodiments, methylsulfonyl.

“Alkoxy” refers to a radical —OR where R is alkyl as defined herein.Examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy.In certain embodiments, an alkoxy group is C₁₋₆ alkoxy, in certainembodiments, C₁₋₅ alkoxy, in certain embodiments, C₁₋₄ alkoxy, incertain embodiments, C₁₋₃ alkoxy, and in certain embodiments, ethoxy ormethoxy.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent aromatic ringsystem. Aryl encompasses 5- and 6-membered carbocyclic aromatic rings,for example, benzene; bicyclic ring systems wherein at least one ring iscarbocyclic and aromatic, for example, naphthalene, indane, andtetralin; and tricyclic ring systems wherein at least one ring iscarbocyclic and aromatic, for example, fluorene. Aryl encompassesmultiple ring systems having at least one carbocyclic aromatic ringfused to at least one carbocyclic aromatic ring, cycloalkyl ring, orheterocycloalkyl ring. For example, aryl includes a phenyl ring fused toa 5- to 7-membered heterocycloalkyl ring containing one or moreheteroatoms selected from N, O, and S. For such fused, bicyclic ringsystems wherein only one of the rings is a carbocyclic aromatic ring,the radical carbon atom may be at the carbocyclic aromatic ring or atthe heterocycloalkyl ring. Examples of aryl groups include groupsderived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene, and the like. In certain embodiments, an aryl group isC₆₋₁₀ aryl, C₆₋₉ aryl, C₆₋₈ aryl, and in certain embodiments, phenyl.Aryl, however, does not encompass or overlap in any way with heteroaryl,separately defined herein.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom is replaced with an aryl group.Examples of arylalkyl groups include benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylalkenyl, or arylalkynyl is used. In certainembodiments, an arylalkyl group is C₇₋₁₆ arylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety of the arylalkyl group is C₁₋₆ and the arylmoiety is C₆₋₁₀, in certain embodiments, an arylalkyl group is C₇₋₁₆arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkylgroup is C₁₋₆ and the aryl moiety is C₆₋₁₀. In certain embodiments anarylalkyl group is C₇₋₉ arylalkyl, wherein the alkyl moiety is C₁₋₃alkyl and the aryl moiety is phenyl. In certain embodiments, anarylalkyl group is C₇₋₁₆ arylalkyl, C₇₋₁₄ arylalkyl, C₇₋₁₂ arylalkyl,C₇₋₁₀ arylalkyl, C₇₋₈ arylalkyl, and in certain embodiments, benzyl.

Bioisosteres are atoms or molecules that fit the broadest definition forisosteres. The concept of bioisosterism is based on the notion thatsingle atom, groups, moieties, or whole molecules, which have chemicaland physical similarities produce similar biological effects. Abioisostere of a parent compound can still be recognized and accepted byits appropriate target, but its functions will be altered as compared tothe parent molecule. Parameters affected with bioisosteric replacementsinclude, for example, size, conformation, inductive and mesomericeffects, polarizability, capacity for electrostatic interactions, chargedistribution, H-bond formation capacity, pKa (acidity), solubility,hydrophobicity, lipophilicity, hydrophilicity, polarity, potency,selectivity, reactivity, or chemical and metabolic stability, ADME(absorption, distribution, metabolism, and excretion). Although commonin pharmaceuticals, carboxyl groups or carboxylic acid functional groups(—CO₂H) in a parent molecule may be replaced with a suitable surrogateor (bio)isostere to overcome chemical or biological shortcomings whileretaining the desired attributes of the parent molecule bearing one ormore carboxyl groups or carboxylic acid functional groups (—CO₂H).Examples of suitable surrogates or (bio)isosteres of carboxyl groups orcarboxylic acid functional groups (—CO₂H) include hydroxamic acids(—CONR¹²OH); boronic acids (—B(OH)(OR¹²), phosphinic acids orderivatives thereof (—PO(OH)R¹²), phosphonic acid or derivatives thereof(—PO(OH)(OR¹²), sulfinic acid (—SOOH), sulfonic acid (—SO₂OH),sulfonamide (—SO₂NHR¹² or —NHSO₂R¹²), sulfonimide or acyl sulfonimide(—SO₂NHCOR¹² or —CONHSO₂R¹²), sulfonylureas (—SO₂NHCONHR¹² or—NHCONHSO₂R¹²), amide (—CONHR¹² or —NHCOR¹²), wherein R¹² in any of theforegoing is selected from hydrogen, C₁₋₆ alkyl, C₁₋₄ fluoroalkyl, C₃₋₆cycloalkyl, and C₆₋₁₀ aryl, acylcyanamide (—CONHCN);2,2,2-trifluoroethan-1-ols (—CH(CF₃)OH), 2,2,2-trifluoromethyl ketonesand hydrates thereof (—COCF₃ and —C(OH)₂CF₃), acidic heterocycles andtheir annular tautomers such as, for example, tetrazole,5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadiazole,5-thioxo-1,2,4-oxadiazole, thiazolidinedione, oxazolidinedione,oxadiazolidinedione, 3-hydroxyisoxazole, 3-hydroxyisothiazole,1-hydroxy-imidazole, 1-hydroxy-pyrazole, 1-hydroxy-triazole,1H-imidazol-2-ol, tetrazole-5-thiol, 3-hydroxyquinolin-2-ones,4-hydroxyquinolin-2-ones, tetronic acid, tetramic acid, mercaptoazolessuch as sulfanyl-1H-imidazole, sulfinyl-1H-imidazole,sulfonyl-1H-imidazole, sulfanyl-1H-triazole, sulfinyl-1H-triazole,sulfonyl-1H-triazole, sulfanyl-1H-1,2,4-triazole,sulfinyl-1H-1,2,4-triazole, sulfonyl-1H-1,2,4-triazole,sulfanyl-1,4-dihydro-1,2,4-triazol-5-one,sulfinyl-1,4-dihydro-1,2,4-triazol-5-one,sulfonyl-1,4-dihydro-1,2,4-triazol-5-one, sulfanyl 1H-tetrazole,sulfanyl 2H-tetrazole, sulfinyl 1H-tetrazole, sulfinyl 2H-tetrazole,sulfonyl 1H-tetrazole, sulfonyl 2H-tetrazole, or sulfonimidamides; andacidic oxocarbocycles or cyclic polyones and their resonance forms suchas, for example, cyclopentane-1,3-diones, squaric acids, squareamides,mixed squaramates, or 2,6-difluorophenols.

“Compounds” of Formula (1) disclosed herein include any specificcompounds within these formulae. Compounds may be identified either bytheir chemical structure and/or chemical name. Compounds are named usingthe ChemDraw Ultra 12.0 (CambridgeSoft, Cambridge, Mass.) nomenclatureprogram. When the chemical structure and chemical name conflict thechemical structure is determinative of the identity of the compound. Thecompounds described herein may comprise one or more stereogenic centersand/or double bonds and therefore may exist as stereoisomers such asdouble-bond isomers (i.e., geometric isomers), enantiomers,diastereomers, or atropisomers. Accordingly, any chemical structureswithin the scope of the specification depicted, in whole or in part,with a relative configuration encompass all possible enantiomers andstereoisomers of the illustrated compounds including thestereoisomerically pure form (e.g., geometrically pure, enantiomericallypure, or diastereomerically pure) and enantiomeric and stereoisomericmixtures. Enantiomeric and stereoisomeric mixtures may be resolved intotheir component enantiomers or stereoisomers using separation techniquesor chiral synthesis techniques well known to the skilled artisan.

Compounds of Formula (1) include optical isomers of compounds of Formula(1), racemates thereof, and other mixtures thereof. In such embodiments,the single enantiomers or diastereomers may be obtained by asymmetricsynthesis or by resolution of the racemates. Resolution of the racematesmay be accomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example a chiral high-pressure liquid chromatography (HPLC)column with chiral stationary phases. In addition, compounds of Formula(1) include (Z)- and (E)-forms (or cis- and trans-forms) of compoundswith double bonds either as single geometric isomers or mixturesthereof.

Compounds of Formula (1) may also exist in several tautomeric formsincluding the enol form, the keto form, and mixtures thereof.Accordingly, the chemical structures depicted herein encompass allpossible tautomeric forms of the illustrated compounds. Compounds mayexist in unsolvated forms as well as solvated forms, including hydratedforms. Certain compounds may exist in multiple crystalline,co-crystalline, or amorphous forms. Compounds of Formula (1) includepharmaceutically acceptable salts thereof, or pharmaceuticallyacceptable solvates of the free acid form of any of the foregoing, aswell as crystalline forms of any of the foregoing

Compounds of Formula (1) are also referred to herein as β-substitutedγ-amino acid derivatives and/or as β-substituted γ-amino acid analogs or(bio)isosteres.

“Chemotherapeutic moiety” refers to a moiety effective in treatingcancer including, any of those disclosed herein. In certain embodiments,a chemotherapeutic moiety may be any suitable chemotherapeutic moiety ofa chemotherapeutic drug known in the art that retains cytotoxic activitywhen bonded either directly or indirectly through a suitable spacingmoiety to a γ-amino acid derivative, γ-amino acid analog, or γ-aminoacid carboxylic acid (bio)isostere as a LAT1 recognition elementprovided by the present disclosure. The conjugate or fusion product ofthe chemotherapeutic moiety with the γ-amino acid derivative, β-aminoacid analog, or β-amino acid carboxylic acid (bio)isostere issimultaneous a selective substrate for the LAT1/4F2hc transporter.

In certain embodiments, the chemotherapeutic moiety, is selected from anitrogen mustard (—N(—CR₂—CR₂—X)₂), a N-monoalkyl or N,N-dialkyltriazene (—N═N—NR₂), a haloacetamide (—NR—CO—CH₂—X), an epoxide(—CROCR—R), an aziridine (—NC₂H₄), a Michael acceptor (—CR═CR-EWG-), asulfonate or a bissulfonate ester (—OSO₂R or ROSO₂—), an N-nitrosourea(—NR—CO—N(NO)R), a bissulfonyl hydrazine (R″SO₂—NR—N(−)—SO₂R′″,—SO₂—NR—NR′—SO₂R′″, or R″SO₂—NR—NR′—SO₂—), a phosphoramidate(—O—P(═O)(N(R)—CH₂—CH₂—X)₂ or —O—P(═O)(N(—CH₂—CH₂—X)₂)₂, and aradionuclide such as, for example, 131-iodine (¹³¹[I]—) or 211-astatine(²¹¹[At]-).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety is a moiety Formula (2a):-A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)  (2a)

wherein,

A is selected from a bond (“-”), oxygen (—O—), sulfur (—S—), amino(—NR¹⁰—), methylene (—CH₂—), methyleneoxy (—CH₂—O—), oxycarbonyl(—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl (—NR¹⁰—C(═O)—),oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl (—S—C(═S)—),aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl (—CH₂—O—C(═O)—),methylenethiocarbonyl (—CH₂—S—C(═O)—), methyleneaminocarbonyl(—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl (—CH₂—O—C(═S)—),methylenethiothiocarbonyl (—CH₂—S—C(═S)—), methyleneaminothiocarbonyl(—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—), methylencarbonyl (—CH₂—C(═O)—),thiocarbonyl (—C(═S)—), and methylenthiocarbonyl (—CH₂—C(═S)—);

Z is selected from a bond (“-”) and oxygen (—O—);

Q is selected from —O⁻ (a negatively charged oxygen atom) that is boundto a positively charged nitrogen atom) and a free electron pair (:),with the proviso that when Q is —O⁻ (a negatively charged oxygen atomthat is bound to a positively charged nitrogen atom), A is selected froma bond (“-”) and methylene (—CH₂—),Z is a bond (“-”), and thechemotherapeutic moiety of Formula (2) is an N-oxide(-A-N⁺(—O⁻)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)₂);

each R¹¹ is independently selected from hydrogen, deuterio, and C₁₋₃alkyl; and

each R⁹ is independently selected from fluoro (—F), chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, whereinR⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl), and (substituted) arylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₆₋₁₀ aryl).

“Cycloalkyl” refers to a saturated or partially unsaturated cyclic alkylradical. In certain embodiments, a cycloalkyl group is C₃₋₆ cycloalkyl,C₃₋₅ cycloalkyl, C₅₋₆ cycloalkyl, cyclopropyl, cyclopentyl, and incertain embodiments, cyclohexyl. In certain embodiments, cycloalkyl isselected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

“Cycloalkylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom is replaced with a cycloalkylgroup as defined herein. Where specific alkyl moieties are intended, thenomenclature cycloalkylalkyl, cycloalkylalkenyl, or cycloalkylalkynyl isused. In certain embodiments, a cycloalkylalkyl group is C₄₋₃₀cycloalkylalkyl, e.g., the alkanyl, alkenyl, or alkynyl moiety of thecycloalkylalkyl group is C₁₋₁₀ and the cycloalkyl moiety of thecycloalkylalkyl moiety is C₃₋₂₀, and in certain embodiments, ancycloalkylalkyl group is C₄₋₂₀ cycloalkylalkyl, e.g., the alkanyl,alkenyl, or alkynyl moiety of the cycloalkylalkyl group is C₁₋₈ and thecycloalkyl moiety of the cycloalkylalkyl group is C₃₋₁₂. In certainembodiments, cycloalkylalkyl is C₄₋₉ cycloalkylalkyl, wherein the alkylmoiety of the cycloalkylalkyl group is C₁₋₃ alkyl, and the cycloalkylmoiety of the cycloalkylalkyl group is C₃₋₆ cycloalkyl. In certainembodiments, a cycloalkylalkyl group is C₄₋₁₂ cycloalkylalkyl, C₄₋₁₀cycloalkylalkyl, C₄₋₈ cycloalkylalkyl, and C₄₋₆ cycloalkylalkyl. Incertain embodiments a cycloalkylalkyl group is cyclopropylmethyl(—CH₂-cyclo-C₃H₅), cyclopentylmethyl (—CH₂-cyclo-C₅H₉), orcyclohexylmethyl (—CH₂-cyclo-C₆H₁₁). In certain embodiments acycloalkylalkyl group is cyclopropylethenyl (—CH═CH-cyclo-C₃H₅),cyclopentylethynyl (—C≡C-cyclo-C₅H₉), or the like.

“Cycloalkylheteroalkyl” by itself or as part of another substituentrefers to a heteroalkyl group in which one or more of the carbon atoms(and certain associated hydrogen atoms) of an alkyl group areindependently replaced with the same or different heteroatomic group orgroups and in which one of the hydrogen atoms bonded to a carbon atom isreplaced with a cycloalkyl group. Where specific alkyl moieties areintended, the nomenclature cycloalkylheteroalkanyl,cycloalkylheteroalkenyl, and cycloalkylheteroalkynyl is used. In certainembodiments of cycloalkylheteroalkyl, the heteroatomic group is selectedfrom —O—, —S—, —NH—, —N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments,the heteroatomic group is selected from —O— and —NH—, and in certainembodiments the heteroatomic group is —O— or —NH—.

“Cycloalkyloxy” refers to a radical —OR where R is cycloalkyl as definedherein. Examples of cycloalkyloxy groups include cyclopropyloxy,cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. In certainembodiments, a cycloalkyloxy group is C₃₋₆ cycloalkyloxy, in certainembodiments, C₃₋₅ cycloalkyloxy, in certain embodiments, C₅₋₆cycloalkyloxy, and in certain embodiments, cyclopropyloxy,cyclobutyloxy, cyclopentyloxy, or cyclohexyloxy.

“Disease” refers to a disease, disorder, condition, or symptom of any ofthe foregoing.

“Fluoroalkyl” refers to an alkyl group as defined herein in which one ormore of the hydrogen atoms is replaced with a fluoro. In certainembodiments, a fluoroalkyl group is C₁₋₆ fluoroalkyl, C₁₋₅ fluoroalkyl,C₁₋₄ fluoroalkyl, C₁₋₃ fluoroalkyl. In certain embodiments, thefluoroalkyl group is pentafluoroethyl (—CF₂CF₃), in certain embodiments,trifluoromethyl (—CF₃).

“Fluoroalkoxy” refers to an alkoxy group as defined herein in which oneor more of the hydrogen atoms is replaced with a fluoro. In certainembodiments, a fluoroalkoxy group is C₁₋₆ fluoroalkoxy, C₁₋₅fluoroalkoxy, C₁₋₄ fluoroalkoxy C₁₋₃ fluoroalkoxy, and in certainembodiments, —OCF₂CF₃ or —OCF₃.

“β-Substituted γ-amino acid derivative” refers to β-substituted γ-aminoacid derivatives having a carboxyl group.

“β-Substituted γ-amino acid analog” refers to β-substituted γ-amino acidderivatives in which the carboxyl group is replaced with a phosphinicacid group, a sulfinic acid group, a 1H-tetrazole, or any of the othersuitable carboxylic acid (bio)isosteres as defined herein, e.g.,3-aminopropylphosphinic acids, 3-aminopropylsulfinic acids, and others.

“Halogen” refers to a fluoro, chloro, bromo, or iodo group.

“Heteroalkoxy” refers to an alkoxy group in which one or more of thecarbon atoms are replaced with a heteroatom. In certain embodiments, theheteroalkoxy group is C₁₋₆ heteroalkoxy, in certain embodiments, C₁₋₅heteroalkoxy, in certain embodiments, C₁₋₄ heteroalkoxy, and in certainembodiments, C₁₋₃ heteroalkoxy. In certain embodiments of heteroalkoxy,the heteroatomic group is selected from —O—, —S—, —NH—, —NR—, —SO₂—, and—SO₂—, in certain embodiments, the heteroatomic group is selected from—O— and —NH—, and in certain embodiments the heteroatomic group is —O—and —NH—. In certain embodiments, a heteroalkoxy group is C₁₋₆heteroalkoxy, C₁₋₅ heteroalkoxy, C₁₋₄ heteroalkoxy, and in certainembodiments C₁₋₃ heteroalkoxy.

“Heteroalkyl” by itself or as part of another substituent refer to analkyl group in which one or more of the carbon atoms (and certainassociated hydrogen atoms) are independently replaced with the same ordifferent heteroatomic group or groups. Examples of heteroatomic groupsinclude —O—, —S—, —NH—, —NR—, —O—O—, —S—S—, ═N—N═, —N═N—, —N═N—NR—,—PR—, —P(O)OR—, —P(O)R—, —POR—, —SO—, —SO₂—, —Sn(R)₂—, and the like,where each R is independently selected from hydrogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₆₋₁₂ aryl, substituted C₆₋₁₂ aryl, C₇₋₁₈arylalkyl, substituted C₇₋₁₈ arylalkyl, C₃₋₇ cycloalkyl, substitutedC₃₋₇ cycloalkyl, C₃₋₇ heterocycloalkyl, substituted C₃₋₇heterocycloalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl, C₆₋₁₂heteroaryl, substituted C₆₋₁₂ heteroaryl, C₇₋₁₈ heteroarylalkyl, andsubstituted C₇₋₁₈ heteroarylalkyl. In certain embodiments, each R isindependently selected from hydrogen and C₁₋₃ alkyl. Reference to, forexample, a C₁₋₆ heteroalkyl, means a C₁₋₆ alkyl group in which at leastone of the carbon atoms (and certain associated hydrogen atoms) isreplaced with a heteroatom. For example, C₁₋₆ heteroalkyl includesgroups having five carbon atoms and one heteroatom, groups having fourcarbon atoms and two heteroatoms, etc. In certain embodiments ofheteroalkyl, the heteroatomic group is selected from —O—, —S—, —NH—,—N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments, the heteroatomicgroup is selected from —O— and —NH—, and in certain embodiments, theheteroatomic group is —O— and —NH—. In certain embodiments, aheteroalkyl group is C₁₋₆ heteroalkyl, C₁₋₅ heteroalkyl, C₁₋₄heteroalkyl, and in certain embodiments C₁₋₃ heteroalkyl.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a parent heteroaromatic ring system.Heteroaryl encompasses multiple ring systems having at least oneheteroaromatic ring fused to at least one other ring, which may bearomatic or non-aromatic. For example, heteroaryl encompasses bicyclicrings in which one ring is heteroaromatic and the second ring is aheterocycloalkyl ring. For such fused, bicyclic heteroaryl ring systemswherein only one of the rings contains one or more heteroatoms, theradical carbon may be at the aromatic ring or at the heterocycloalkylring. In certain embodiments, when the total number of N, S, and O atomsin the heteroaryl group exceeds one, the heteroatoms may or may not beadjacent to one another. In certain embodiments, the total number ofheteroatoms in the heteroaryl group is not more than two. In certainembodiments of heteroaryl, the heteroatomic group is selected from —O—,—S—, —NH—, —N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments, theheteroatomic group is selected from —O— and —NH—, and in certainembodiments the heteroatomic group is —O— or —NH—. In certainembodiments, a heteroaryl group is selected from C₅₋₁₀ heteroaryl, C₅₋₉heteroaryl, C₅₋₈ heteroaryl, C₅₋₇ heteroaryl, C₅₋₆ heteroaryl, and incertain embodiments, is C₅ heteroaryl or C₆ heteroaryl.

Examples of heteroaryl groups include groups derived from acridine,arsindole, carbazole, α-carboline, chromane, chromene, cinnoline, furan,imidazole, indazole, indole, indoline, indolizine, isobenzofuran,isochromene, isoindole, isoindoline, isoquinoline, isothiazole,isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,thiazolidine, oxazolidine, and the like. In certain embodiments,heteroaryl groups are those derived from thiophene, pyrrole,benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,oxazole, or pyrazine. For example, in certain embodiments, heteroaryl isC₅ heteroaryl and is selected from furyl, thienyl, pyrrolyl, imidazolyl,pyrazolyl, isothiazolyl, isoxazolyl. In certain embodiments, heteroarylis C₆ heteroaryl, and is selected from pyridinyl, pyrazinyl,pyrimidinyl, and pyridazinyl.

“Heteroarylalkyl” refers to an arylalkyl group in which one of thecarbon atoms (and certain associated hydrogen atoms) is replaced with aheteroatom. In certain embodiments, a heteroarylalkyl group is C₆₋₁₆heteroarylalkyl, C₆₋₁₄ heteroarylalkyl, C₆₋₁₂ heteroarylalkyl, C₆₋₁₀heteroarylalkyl, C₆₋₈ heteroarylalkyl, C₇ heteroarylalkyl, and incertain embodiments, C₆ heteroarylalkyl. In certain embodiments ofheteroarylalkyl, the heteroatomic group is selected from —O—, —S—, —NH—,—N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments, the heteroatomicgroup is selected from —O— and —NH—, and in certain embodiments theheteroatomic group is —O— or —NH—.

“Heterocycloalkyl” by itself or as part of another substituent refers toa saturated or unsaturated cyclic alkyl radical in which one or morecarbon atoms (and certain associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom; or to a parent aromaticring system in which one or more carbon atoms (and certain associatedhydrogen atoms) are independently replaced with the same or differentheteroatom such that the ring system violates the Huckel-rule. Examplesof heteroatoms to replace the carbon atom(s) include N, P, O, S, and Si.Examples of heterocycloalkyl groups include groups derived fromepoxides, azirines, thiiranes, imidazolidine, morpholine, piperazine,piperidine, pyrazolidine, pyrrolidine, quinuclidine, and the like. Incertain embodiments, heterocycloalkyl is C₅ heterocycloalkyl and isselected from pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl,imidazolidinyl, oxazolidinyl, thiazolidinyl, doxolanyl, and dithiolanyl.In certain embodiments, heterocycloalkyl is C₆ heterocycloalkyl and isselected from piperidinyl, tetrahydropyranyl, piperizinyl, oxazinyl,dithianyl, and dioxanyl. In certain embodiments a heterocycloalkyl groupis C₃₋₆ heterocycloalkyl, C₃₋₅ heterocycloalkyl, C₅₋₆ heterocycloalkyl,and in certain embodiments, C₅ heterocycloalkyl or C₆ heterocycloalkyl.In certain embodiments of heterocycloalkyl, the heteroatomic group isselected from —O—, —S—, —NH—, —N(—CH₃)—, —SO—, and —SO₂—, in certainembodiments, the heteroatomic group is selected from —O— and —NH—, andin certain embodiments the heteroatomic group is —O— or —NH—.

“Heterocycloalkylalkyl” refers to a cycloalkylalkyl group in which oneor more carbon atoms (and certain associated hydrogen atoms) of thecycloalkyl ring are independently replaced with the same or differentheteroatom. In certain embodiments, the heterocycloalkylalkyl is C₄₋₁₂heterocycloalkylalkyl, C₄₋₁₀ heterocycloalkylalkyl, C₄₋₈heterocycloalkylalkyl, C₄₋₆ heterocycloalkylalkyl, C₆₋₇heterocycloalkylalkyl, and in certain embodiments, C₆heterocycloalkylalkyl or C₇ heterocycloalkylalkyl. In certainembodiments of heterocycloalkylalkyl, the heteroatomic group is selectedfrom —O—, —S—, —NH—, —N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments,the heteroatomic group is selected from —O— and —NH—, and in certainembodiments the heteroatomic group is —O— or —NH—.

“Mesyl” refers to the group —OS(O)₂Me or -OMs.

“Parent aromatic ring system” refers to an unsaturated cyclic orpolycyclic ring system having a cyclic conjugated π (pi) electron systemwith 4n+2 electrons (Hückel rule). Included within the definition of“parent aromatic ring system” are fused ring systems in which one ormore of the rings are aromatic and one or more of the rings aresaturated or unsaturated, such as, for example, fluorene, indane,indene, phenalene, etc. Examples of parent aromatic ring systems includeaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like.

“Parent heteroaromatic ring system” refers to an aromatic ring system inwhich one or more carbon atoms (and any associated hydrogen atoms) areindependently replaced with the same or different heteroatom in such away as to maintain the continuous 7-electron system characteristic ofaromatic systems and a number of 7-electrons corresponding to the Hückelrule (4n+2). Examples of heteroatoms to replace the carbon atoms includeN, P, O, S, and Si, etc. Specifically included within the definitionof“parent heteroaromatic ring systems” are fused ring systems in whichone or more of the rings are aromatic and one or more of the rings aresaturated or unsaturated, such as, for example, arsindole, benzodioxan,benzofuran, chromane, chromene, indole, indoline, xanthene, etc.Examples of parent heteroaromatic ring systems include arsindole,carbazole, O-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,thiazolidine, oxazolidine, and the like.

“Patient” refers to a mammal, for example, a human. The term “patient”is used interchangeably with “subject.”

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound, whichpossesses the desired pharmacological activity of the parent compound.Such salts include acid addition salts, formed with inorganic acids andone or more protonable functional groups such as primary, secondary, ortertiary amines within the parent compound or formed with an organicacids. Examples for inorganic acids are hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, and the like.Examples of organic acids include acetic acid, propionic acid, hexanoicacid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lacticacid, malonic acid, succinic acid, malic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonicacid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, lauryl sulfuric acid, gluconic acid, glutamic acid,hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, andthe like. In certain embodiments, a pharmaceutically acceptable salt isa salt formed when one or more acidic protons present in the parentcompound are replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion, or combinations thereof; orcoordinates with an organic base such as ethanolamine, diethanolamine,triethanolamine, N-methylglucamine, and the like. In certainembodiments, a pharmaceutically acceptable salt is the hydrochloridesalt. In certain embodiments, a pharmaceutically acceptable salt is thesodium salt. In certain embodiments wherein a compound has two or moreionizable groups, a pharmaceutically acceptable salt comprises one ormore counterions, such as a bi-salt, for example, a dihydrochloridesalt.

The term “pharmaceutically acceptable salt” includes hydrates and othersolvates, as well as salts in crystalline or non-crystalline form. Wherea particular pharmaceutically acceptable salt is disclosed, it isunderstood that the particular salt (e.g., a hydrochloride salt) is anexample of a salt, and that other salts may be formed using techniquesknown to one of skill in the art. Additionally, one of skill in the artwould be able to convert the pharmaceutically acceptable salt to thecorresponding compound, free base and/or free acid, using techniquesgenerally known in the art. See also: Stahl and Wermuth (Editors),Handbook of Pharmaceutical Salts, Wiley-VCH, Weinheim, Germany, 2008.

“Pharmaceutically acceptable vehicle” refers to a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable adjuvant, apharmaceutically acceptable excipient, a pharmaceutically acceptablecarrier, or a combination of any of the foregoing with which a compoundprovided by the present disclosure may be administered to a patient andwhich does not destroy the pharmacological activity thereof and which isnon-toxic when administered in doses sufficient to provide atherapeutically effective amount of the compound.

“Pharmaceutical composition” refers to a compound of Formula (1) or apharmaceutically acceptable salt thereof and at least onepharmaceutically acceptable vehicle, with which the compound of Formula(1) or a pharmaceutically acceptable salt thereof is administered to apatient. Pharmaceutically acceptable vehicles are known in the art.

“Solvate” refers to a molecular complex of a compound with one or moresolvent molecules in a stoichiometric or non-stoichiometric amount. Suchsolvent molecules are those commonly used in the pharmaceutical arts,which are known to be innocuous to a patient, e.g., water, ethanol, andthe like. A molecular complex of a compound or moiety of a compound anda solvent can be stabilized by non-covalent intra-molecular forces suchas, for example, electrostatic forces, van der Waals forces, or hydrogenbonds. The term “hydrate” refers to a solvate in which the one or moresolvent molecules is water.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s). Incertain embodiments, each substituent is independently selected fromhalogen, —OH, —CN, —CF₃, —OCF₃, ═O, —NO₂, C₁₋₆ alkoxy, C₁₋₆ alkyl,—COOR, —NR₂, and —CONR₂; wherein each R is independently selected fromhydrogen and C₁₋₆ alkyl. In certain embodiments, each substituent isindependently selected from halogen, —NH₂, —OH, C₁₋₃ alkoxy, and C₁₋₃alkyl, trifluoromethoxy, and trifluoromethyl. In certain embodiments,each substituent is independently selected from —OH, methyl, ethyl,trifluoromethyl, methoxy, ethoxy, and trifluoromethoxy. In certainembodiments, each substituent is selected from C₁₋₃ alkyl, ═O, C₁₋₃alkyl, C₁₋₃ alkoxy, and phenyl. In certain embodiments, each substituentis selected from —OH, —NH₂, C₁₋₃ alkyl, and C₁₋₃ alkoxy.

“Treating” or “treatment” of a disease refers to arresting orameliorating a disease or at least one of the clinical symptoms of adisease or disorder, reducing the risk of acquiring a disease or atleast one of the clinical symptoms of a disease, reducing thedevelopment of a disease or at least one of the clinical symptoms of thedisease or reducing the risk of developing a disease or at least one ofthe clinical symptoms of a disease. “Treating” or “treatment” alsorefers to inhibiting the disease, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both, and to inhibiting atleast one physical parameter or manifestation that may or may not bediscernible to the patient. In certain embodiments, “treating” or“treatment” refers to delaying the onset of the disease or at least oneor more symptoms thereof in a patient who may be exposed to orpredisposed to a disease or disorder even though that patient does notyet experience or display symptoms of the disease.

“Therapeutically effective amount” refers to the amount of a compoundthat, when administered to a subject for treating a disease, or at leastone of the clinical symptoms of a disease, is sufficient to affect suchtreatment of the disease or symptom thereof. The “therapeuticallyeffective amount” may vary depending, for example, on the compound, thedisease and/or symptoms of the disease, severity of the disease and/orsymptoms of the disease or disorder, the age, weight, and/or health ofthe patient to be treated, and the judgment of the prescribingphysician. An appropriate amount in any given instance may beascertained by those skilled in the art or capable of determination byroutine experimentation.

“Therapeutically effective dose” refers to a dose that provideseffective treatment of a disease or disorder in a patient. Atherapeutically effective dose may vary from compound to compound, andfrom patient to patient, and may depend upon factors such as thecondition of the patient and the route of delivery. A therapeuticallyeffective dose may be determined in accordance with routinepharmacological procedures known to those skilled in the art.

“Triflyl” refers to the group —OS(O)₂CF₃ or -OTf.

Reference is now made in detail to certain embodiments of compounds,compositions, and methods. The disclosed embodiments are not intended tobe limiting of the claims. To the contrary, the claims are intended tocover all alternatives, modifications, and equivalents.

LAT1/4F2hc Transporter

The GenBank accession number for human LAT1/4F2hc isNP_003477/NP_002385. Unless otherwise apparent from the context,reference to a transporter such as LAT1/4F2hc (as well as othertransporters disclosed herein) includes the amino acid sequencedescribed in or encoded by the GenBank reference number, and, allelic,cognate and induced variants and fragments thereof retaining essentiallythe same transporter activity. Usually such variants show at least 90%sequence identity to the exemplary Genbank nucleic acid or amino acidsequence. Allelic variants at the DNA level are the result of geneticvariation between individuals of the same species. Some allelic variantsat the DNA level that cause substitution, deletion or insertion of aminoacids in proteins encoded by the DNA result in corresponding allelicvariation at the protein level. Cognate forms of a gene refer tovariation between structurally and functionally related genes betweenspecies. For example, the human gene showing the greatest sequenceidentity and closest functional relationship to a mouse gene is thehuman cognate form of the mouse gene.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm enables calculation of the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated program parameters. Optimal alignment ofsequences for comparison may be conducted by methods known to thoseskilled in the art.

Compounds

In certain embodiments, anti-cancer agents provided by the presentdisclosure are compounds of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein:

at least one of R¹ and R⁵ is independently selected from halogen,—N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(OR¹⁰)(R¹⁰), —NO₂, —NO, —N(R¹⁰)(S(═O)R¹⁰),—N(R¹⁰)(S(═O)₂R¹⁰), —N(R¹⁰)(C(O)R¹⁰), —N(R¹⁰)(C(O)OR¹⁰),—N(R¹⁰)(C(O)N(R¹⁰)₂, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, —SH, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, —S(O)N(R¹⁰)₂,—S(O)₂N(R¹⁰)₂, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, C₃₋₆cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyloxy, substitutedC₃₋₆ cycloalkyloxy, C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂cycloalkylalkyl, C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, C₇₋₁₆ arylalkyl,substituted C₇₋₁₆ arylalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ heteroalkoxy, substituted C₁₋₆ heteroalkoxy, C₃₋₆heterocycloalkyl, substituted C₃₋₆ heterocycloalkyl, C₄₋₁₂heterocycloalkylalkyl, substituted C₄₋₁₂ heterocycloalkylalkyl, C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₆₋₁₆ heteroarylalkyl, andsubstituted C₆₋₁₆ heteroarylalkyl;

one of R¹, R², R³, R⁴, and R⁵ comprises a chemotherapeutic moiety;

each of the other of R¹, R², R³, R⁴, and R⁵ is independently selectedfrom hydrogen, deuterio, halogen, —OH, —N(R¹⁰)₂, —NO₂, —NO, —CN,—COOR¹⁰, —CON(R¹⁰)₂, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₆ cycloalkyl,substituted C₃₋₆ cycloalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, C₁₋₆ heteroalkoxy,substituted C₁₋₆ heteroalkoxy, C₄₋₈ cycloalkylalkyl, and C₄₋₈cycloalkylheteroalkyl;

R⁶ is selected from a carboxylic acid (—COOH), a carboxylic acid analog,and a carboxylic acid (bio)isostere;

each R⁷ is independently selected from hydrogen, deuterio, halogen,hydroxyl, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, and phenyl; or two R⁷together with the carbon to which they are bonded form a ring selectedfrom a C₃₋₆ cycloalkyl ring and a C₃₋₆ heterocycloalkyl ring;

R⁸ is selected from hydrogen, deuterio, halogen, C₁₋₆ alkyl, substitutedC₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl, C₁₋₆ alkoxy,substituted C₁₋₆ alkoxy, C₁₋₆ heteroalkoxy, substituted C₁₋₆heteroalkoxy, C₃₋₆ cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆cycloalkyloxy, substituted C₃₋₆ cycloalkyloxy, —OH, —COOR¹⁰, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₆ cycloalkyl, and phenyl;

each R¹⁰ is independently selected from hydrogen, C₁₋₄ alkyl, and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring;

L is —(X)_(a)—, wherein each X is independently selected from a bond(“-”), —C(R¹⁶)₂—, wherein each R¹⁶ is independently selected fromhydrogen, deuterio, halogen, hydroxyl, C₁₋₄ alkyl, and C₁₋₄ alkoxy, ortwo R¹⁶ together with the carbon to which they are bonded form a C₃₋₆cycloalkyl ring or a C₃₋₆ heterocycloalkyl ring, —O—, —S—, —SO—, —SO₂—,—CO—, and —N(R¹⁷)— wherein R¹⁷ is selected from hydrogen and C₁₋₄ alkyl;

a is selected from 0, 1, 2, 3, and 4; and

each substituent is independently selected from halogen, —OH, —NH₂,—N(R¹⁰)₂, —NO₂, —CF₃, ═O (oxo), C₁₋₃ alkyl, C₁₋₃ alkoxy, and phenyl;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl.

In certain embodiments in compounds of Formula (1), R¹ comprises achemotherapeutic moiety, R² comprises a chemotherapeutic moiety, R³comprises a chemotherapeutic moiety, R⁴ comprises a chemotherapeuticmoiety, and in certain embodiments, R⁵ comprises a chemotherapeuticmoiety.

In certain embodiments of a compound of Formula (1), a chemotherapeuticmoiety may be any suitable chemotherapeutic moiety of a chemotherapeuticdrug known in the art that retains cytotoxic activity when bonded eitherdirectly or indirectly through a suitable spacing moiety, e.g., an arylring and a linker L, to na γ-amino acid derivative, γ-amino acid analog,or γ-amino acid carboxylic acid (bio)isostere as a LAT1 recognitionelement provided by the present disclosure. The conjugate or fusionproduct of the chemotherapeutic moiety with the γ-amino acid derivative,γ-amino acid analog, or γ-amino acid carboxylic acid (bio)isostere issimultaneous a selective substrate for the LAT1/4F2hc transporter.

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety, is selected from a nitrogen mustard(—N(—CR₂—CR₂—X)₂), a N-monoalkyl or N,N-dialkyl triazene (—N═N—NR₂), ahaloacetamide (—NR—CO—CH₂—X), an epoxide (—CROCR—R), an aziridine(—NC₂H₄), a Michael acceptor (—CR═CR-EWG-), a sulfonate or abissulfonate ester (—OSO₂R or ROSO₂—), an N-nitrosourea (—NR—CO—N(NO)R),a bissulfonyl hydrazine (R″SO₂—NR—N(−)—SO₂R′″, —SO₂—NR—NR′—SO₂R′″, orR″SO₂—NR—NR′—SO₂—), a phosphoramidate (—O—P(═O)(N(R)—CH₂—CH₂—X)₂ or—O—P(═O)(N(—CH₂—CH₂—X)₂)₂, and a radionuclide such as, for example,131-iodine (¹³¹[I]—) or 211-astatine (²¹¹[At]-).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety is a moiety Formula (2):

wherein,

A is selected from a bond (“-”), oxygen (—O—), sulfur (—S—), amino(—NR¹⁰—), methylene (—CH₂—), methyleneoxy (—CH₂—O—), oxycarbonyl(—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl (—NR¹⁰—C(═O)—),oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl (—S—C(═S)—),aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl (—CH₂—O—C(═O)—),methylenethiocarbonyl (—CH₂—S—C(═O)—), methyleneaminocarbonyl(—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl (—CH₂—O—C(═S)—),methylenethiothiocarbonyl (—CH₂—S—C(═S)—), methyleneaminothiocarbonyl(—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—), methylencarbonyl (—CH₂—C(═O)—),thiocarbonyl (—C(═S)—), and methylenthiocarbonyl (—CH₂—C(═S)—);

Z is selected from a bond (“-”) and oxygen (—O—);

Q is selected from —O⁻ (a negatively charged oxygen atom) that is boundto a positively charged nitrogen atom) and a free electron pair (:),with the proviso that when Q is —O⁻ (a negatively charged oxygen atomthat is bound to a positively charged nitrogen atom), A is selected froma bond (“-”) and methylene (—CH₂—),Z is a bond (“-”), and thechemotherapeutic moiety of Formula (2) is an N-oxide(-A-N⁺(—O⁻)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)₂);

each R¹¹ is independently selected from hydrogen, deuterio, and C₁₋₃alkyl; and

each R⁹ is independently selected from fluoro (—F), chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, whereinR⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl), and (substituted) arylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is C₆₋₁₀ aryl).

In certain embodiments, a chemotherapeutic moiety of Formula (2) isselected from the structure-A-N(—Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹) and-A-N⁺(—O⁻)(—C(R¹¹)₂−C(R¹¹)₂—R⁹)₂,

wherein A is selected from a bond (“-”), methylene (—CH₂—), oxygen(—O—), methyleneoxy (—CH₂—O—), oxycarbonyl (—O—C(═O)—),methyleneoxycarbonyl (—CH₂—O—C(═O)—), carbonyl (—C(═O)—), andmethylenecarbonyl (—CH₂—C(═O)—);

each R¹¹ is independently selected from hydrogen and deuterio; and

each R⁹ is independently selected from fluoro (—F), chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, whereinR⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl), and (substituted) arylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₆₋₁₀ aryl).

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹, wherein A isa bond (“-”); Q is a free electron pair (:); Z is a bond (“-”); each R¹¹is independently selected from hydrogen and deuterio; and each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I), alkylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), and C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl); and the chemotherapeutic moiety is—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n are independentlyselected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais methylene (—CH₂—); Q is a free electron pair (:); Z is a bond (“-”);each R¹¹ is independently selected from hydrogen and deuterio; and eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), andC₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected fromC₁₋₄ (per)fluoroalkyl); and the chemotherapeutic moiety is—CH₂—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais a bond (“-”); Q is a negatively charged oxygen (—O—); Z is a bond(“-”); each R¹¹ is independently selected from hydrogen and deuterio;and each R⁹ is independently selected from chloro (—Cl), bromo (—Br),iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from C₁₋₄ (per)fluoroalkyl); and the chemotherapeutic moiety is—N⁺(—O⁻)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais methylene (—CH₂—); Q is a negatively charged oxygen (—O—); Z is abond (“-”); each R¹¹ is independently selected from hydrogen anddeuterio; and each R⁹ is independently selected from chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰,wherein R⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl); and thechemotherapeutic moiety is—CH₂—N⁺(—O⁻)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais a bond (“-”); Q is a free electron pair (:); Z is oxygen; each R¹¹ isindependently selected from hydrogen and deuterio; and each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I), alkylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), and C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl); and the chemotherapeutic moiety is—N(—O—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹),wherein m and n are independently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais methylene (—CH₂—); Q is a free electron pair (:); Z is oxygen; eachR¹¹ is independently selected from hydrogen and deuterio; and each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I), alkylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), anf C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl); and the chemotherapeutic moiety is—CH₂—N(—O—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹),wherein m and n are independently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais oxygen (—O—); Q is a free electron pair (:); Z is a bond (“-”); eachR¹¹ is independently selected from hydrogen and deuterio; and each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I), alkylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), and C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl) and the chemotherapeutic moiety is—O—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais methyleneoxy (—CH₂—O—); Q is a free electron pair (:); Z is a bond(“-”); each R¹¹ is independently selected from hydrogen and deuterio;and each R⁹ is independently selected from chloro (—Cl), bromo (—Br),iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from C₁₋₄ (per)fluoroalkyl); and the chemotherapeutic moiety is—CH₂—O—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais a carbonyl (—CO—), Q is a free electron pair (:), Z is a bond (“-”),each R¹¹ is independently selected from hydrogen and deuterio; and eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl),C₁₋₄ (per)fluoroalklyl sulfonate ((—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ (per)fluoroalkyl) and the chemotherapeutic moiety is(—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂), wherein m and n areindependently selected integers of 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais methylenecarbonyl (—CH₂—CO—); Q is a free electron pair (:); Z is abond (“-”); each R¹¹ is independently selected from hydrogen anddeuterio; and each R⁹ is independently selected from chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰,wherein R⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl); and thechemotherapeutic moiety is —CH₂—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂,wherein m and n are independently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais oxycarbonyl (—O—CO—); Q is a free electron pair (:); Z is a bond(“-”); each R¹¹ is independently selected from hydrogen and deuterio;and each R⁹ is independently selected from chloro (—Cl), bromo (—Br),iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from C₁₋₄ (per)fluoroalkyl); and the chemotherapeutic moiety is—O—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais a methyleneoxycarbonyl (—CH₂—O—CO—); each R¹¹ is independentlyselected from hydrogen and deuterio; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), alkyl sulfonate(—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), and C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl); and the chemotherapeutic moiety is—CH₂—O—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂,wherein m and n are independently selected from 0, 1, and 2; and each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—N⁺(—O⁻)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—N⁺(—O⁻)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—N(—O—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹),wherein m and n are independently selected from 0, 1, and 2; and each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—N(—O—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹),wherein m and n are independently selected from 0, 1, and 2; and each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—O—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected integers of 0, 1, and 2; and each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—O—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—O—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—O—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, wherein eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, whereineach R⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo(—I), methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy(—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),wherein each R⁹ is independently selected from chloro (—Cl), bromo(—Br), iodo (—I), methylsulfonyloxy (—OSO₂CH₃), andtrifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),wherein each R⁹ is independently selected from chloro (—Cl), bromo(—Br), iodo (—I), methylsulfonyloxy (—OSO₂CH₃), andtrifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —O—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—O—N(—CH₂—CH₂—R⁹)₂, wherein eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—CO—N(—CH₂—CH₂—R⁹)₂, wherein eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —O—CO—N(—CH₂—CH₂—R⁹)₂, wherein m and nare independently selected from 0, 1, and 2, and each of R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, whereineach R⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo(—I), methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy(—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), R⁶ is selected fromcarboxylic acid (—COOH), hydroxamic acids (—CONR¹²OH), boronic acids(—B(OH)(OR¹²), phosphinic acids or derivatives thereof (—PO(OH)R¹²), andphosphonic acid or derivatives thereof (—PO(OH)(OR¹²)), sulfinic acid(—SOOH), sulfonic acid (—SO₂OH), sulfonamide (—SO₂NHR¹² or —NHSO₂R¹²),sulfonimide or acyl sulfonimide (—SO₂NHCOR¹² or —CONHSO₂R¹²),sulfonylureas (—SO₂NHCONHR¹² or —NHCONHSO₂R¹²), amide (—CONHR¹² or—NHCOR¹²), acylcyanamide (—CONHCN), 2,2,2-trifluoroethan-1-ols(—CH(CF₃)OH), 2,2,2-trifluoromethyl ketones and hydrates thereof (—COCF₃and —C(OH)₂CF₃), acidic heterocycles and annular tautomers of any of theforegoing, and acidic oxocarbocycles or cyclic polyones and resonanceforms of any of the foregoing; wherein R¹² is selected from hydrogen,C₁₋₆ alkyl, C₁₋₄ fluoroalkyl, C₃₋₆ cycloalkyl, and C₆₋₁₀ aryl.

In certain embodiments of a compound of Formula (1), the acidicheterocycle and annular tautomers thereof is selected from 1H-tetrazole,5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadiazole,5-thioxo-1,2,4-oxadiazole, thiazolidinedione, oxazolidinedione,oxadiazolidinedione, 3-hydroxyisoxazole, 3-hydroxyisothiazole,1-hydroxy-imidazole, 1-hydroxy-pyrazole, 1-hydroxy-triazole,1H-imidazol-2-ol, tetrazole-5-thiol, 3-hydroxyquinolin-2-one,4-hydroxyquinolin-2-ones, tetronic acid, tetramic acid, mercaptoazolessuch as sulfanyl-1H-imidazole, sulfinyl-1H-imidazole,sulfonyl-1H-imidazole, sulfanyl-1H-triazole, sulfinyl-1H-triazole,sulfonyl-1H-triazole, sulfanyl-1H-1,2,4-triazole,sulfinyl-1H-1,2,4-triazole, sulfonyl-1H-1,2,4-triazole,sulfanyl-1,4-dihydro-1,2,4-triazol-5-one,sulfinyl-1,4-dihydro-1,2,4-triazol-5-one,sulfonyl-1,4-dihydro-1,2,4-triazol-5-one, sulfanyl 1H-tetrazole,sulfanyl 2H-tetrazole, sulfinyl 1H-tetrazole, sulfinyl 2H-tetrazole,sulfonyl 1H-tetrazole, sulfonyl 2H-tetrazole, and sulfonimidamide.

In certain embodiments of a compound of Formula (1), the acidicoxocarbocycle or cyclic polyone and resonance forms thereof is selectedfrom cyclopentane-1,3-dione, squaric acid, squareamide, mixedsquaramate, and 2,6-difluorophenol.

In certain embodiments of a compound of Formula (1), R⁶ is selected from—COOH, —S(O)OH, —SO₂OH, —P(O)(OH)R¹², —P(O)(OH)(OR¹²), —SO₂NHR¹²,—NHSO₂R¹², —SO₂NHCOR¹², —CONHSO₂R¹², —SO₂NHCONHR¹², —CONHCN,1H-tetrazol-yl, 5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadiazole,5-thioxo-1,2,4-oxadiazole, thiazolidinedione, oxazolidinedione,oxadiazolidinedione, 3-hydroxyisoxazole, 3-hydroxyisothiazole,cyclopentane-1,3-dione, squaric acid, squareamide, and mixed squaramate;wherein R¹² is selected from hydrogen, C₁₋₄ alkyl, and C₃₋₅ cycloalkyl.

In certain embodiments of a compound of Formula (1), R⁶ is selected from—COOH, —S(O)OH, —P(O)(OH)H, —CONHSO₂CH₃, —CONHSO₂CF₃, —SO₂NHCOCH₃,—SO₂NHCOCF₃, —NHSO₂CH₃, —NHSO₂CF₃, 1H-tetrazol-yl,5-oxo-1,2,4-oxadiazole-yl, 5-oxo-1,2,4-thiadiazole-yl,5-thioxo-1,2,4-oxadiazole-yl, thiazolidinedione-yl, oxazolidinedione-yl,oxadiazolidinedione-yl, 3-hydroxyisoxazole-yl, 3-hydroxyisothiazole-yl,tetronic acid-yl, tetramic acid-yl, and cyclopentane-1,3-dione-yl.

In certain embodiments of a compound of Formula (1), R⁶ is selected from—COOH, —S(O)OH, —P(O)(OH)H, —CONHSO₂CH₃, —CONHSO₂CF₃, —SO₂NHCOCH₃,—SO₂NHCOCH₃, —SO₂NHCOCF₃, —NHSO₂CF₃, —NHSO₂CF₃, and 1H-tetrazol-5-yl.

In certain embodiments of a compound of Formula (1), R⁶ is selected from—COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazol-yl.

In certain embodiments of a compound of Formula (1), R⁶ is —COOH.

In certain embodiments of a compound of Formula (1), each R⁷ isindependently selected from hydrogen, deuterio, halogen, hydroxyl, andC₁₋₄ alkyl, or two germinal R⁷ together with the carbon atom to whichthey are bonded form a C₃₋₅ cycloalkyl ring.

In certain embodiments of a compound of Formula (1), each R⁷ isindependently selected from hydrogen, deuterio, fluoro, hydroxyl,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl, ortwo germinal R⁷ together with the carbon atom to which they are bondedform a cyclopropyl ring or a cyclobutyl ring.

In certain embodiments of a compound of Formula (1), each R⁷ isindependently selected from hydrogen, deuterio, fluoro, hydroxyl, andmethyl.

In certain embodiments of a compound of Formula (1), each R⁷ isindependently selected from hydrogen and deuterio.

In certain embodiments of a compound of Formula (1), each R⁷ ishydrogen.

In certain embodiments of a compound of Formula (1), R⁸ is selected fromhydrogen, deuterio, halogen, hydroxyl, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, and cyclopropyl.

In certain embodiments of a compound of Formula (1), R⁸ is selected fromhydrogen, deuterio, halogen, hydroxyl, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, trifluoromethyl, methoxy, ethoxy,isopropoxy, trifluoromethoxy, and cyclopropyl.

In certain embodiments of a compound of Formula (1), R⁸ is selected fromhydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, tert-butyl,hydroxyl, methoxy, ethoxy, isopropoxy, trifluoromethyl, andtrifluoromethoxy.

In certain embodiments of a compound of Formula (1), R⁸ is methyl.

In certain embodiments of a compound of Formula (1), R⁸ is hydrogen.

In certain embodiments of a compound of Formula (1), each R¹⁰ isindependently selected from hydrogen and C₁₋₄ alkyl, or two R¹⁰ togetherwith the nitrogen atom to which they are bonded form a 3- to 5-memberedheterocycle.

In certain embodiments of a compound of Formula (1), L is (—X—)_(a)wherein a is selected from 0, 1, 2, 3, and 4 and X is selected fromoxygen (—O—), sulfur (—S—), sulfinyl (—SO—), sulfonyl (—SO₂—), carbonyl(—CO—), —C(R¹⁶)₂— wherein R¹⁶ is independently selected from hydrogen,deuterio, halogen, hydroxyl, C₁₋₄ alkyl, and amino (—NR¹⁷—), wherein R¹⁷is selected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1), a is 1, X and L arethe same, and each of X and L is selected from a bond (“-”), methylene(—CH₂—), fluoromethylene (—CFH—), difluoromethylene (—CF₂—),hydroxymethylene (—C(OH)H—), ethane-1,1-diyl (—CHCH₃—), propane-2,2-diyl(—C(CH₃)₂—), propane-1,1-diyl (—CH(CH₂—CH₃)—), oxygen (—O—), sulfur(—S—), sulfinyl (—SO—), sulfonyl (—SO₂—), carbonyl (—CO—), and amino(—NR¹⁷—), wherein R¹⁷ is selected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1), a is 2, each X ismethylene (—CH₂—), and L is ethane-1,2-diyl (—CH₂—CH₂—); one X ismethylene (—CH₂—) and one X is ethane-1,1-diyl (—CHCH₃—) and L ispropane-1,2-diyl (—CH₂—CHCH₃—); one X is ethane-1,1-diyl (—CHCH₃—) andone X is methylene (—CH₂—) and L is propane-1,2-diyl (—CHCH₃—CH₂—); oneX is methylene (—CH₂—) and one X is hydroxymethylene (—CHOH—) and L ishydroxyethane-1,2-diyl (—CH₂—CHOH—); one X is hydroxymethylene (—CHOH—)and one X is methylene (—CH₂—) and L is hydroxyethane-1,2-diyl(—CHOH—CH₂—); one X is methylene (—CH₂—) and one X is fluoromethylene(—CFH—), and L is fluoroethane-1,2-diyl (—CH₂—CHF—); one X isfluoromethylene (—CFH—), and one X is methylene (—CH₂—) and L isfluoroethane-1,2-diyl (—CHF—CH₂—); one X is methylene (—CH₂—) and one Xis difluoromethylene (—CF₂—), and L is difluoroethane-1,2-diyl(—CH₂—CF₂—); one X is difluoromethylene (—CF₂—), and one X is methylene(—CH₂—) and L is difluoroethane-1,2-diyl (—CF₂—CH₂—); one X is carbonyl(—CO—) and one X is amino (—NR¹⁷—) and L is carbonyl amino (—CO—NR¹⁷—);one X is amino (—NR¹⁷—) and one X is carbonyl (—CO—) and L is aminocarbonyl (—NR¹⁷—CO—); one X is methylene (—CH₂—) and one X is amino(—NR¹⁷—) and L is methyleneamino (—CH₂—NR¹⁷—); one X is amino (—NR¹⁷—)and one X is methylene (—CH₂—) and L is aminomethylene (—NR¹⁷—CH₂—); oneX is methylene (—CH₂—) and one X is oxygen (—O—) and L is methyleneoxy(—CH₂—O—); one X is oxygen (—O—) and one X is methylene (—CH₂—) and L isoxymethylen (—O—CH₂—); one X is methylene (—CH₂—) and one X is sulfur(—S—) and L is methylenethiyl (—CH₂—S—); one X is sulfur (—S—) and one Xis methylene (—CH₂—) and L is thiylmethylene (—S—CH₂—); one X ismethylene (—CH₂—) and one X is sulfinyl (—SO—) and L ismethylenesulfinyl (—CH₂—SO—); one X is sulfinyl (—SO—) and one X ismethylene (—CH₂—) and L is sulfinylmethylene (—SO—CH₂—); one X ismethylene (—CH₂—) and one X is sulfonyl (—SO₂—) and L ismethylenesulfonyl (—CH₂—SO₂—); one X is sulfonyl (—SO₂—) and one X ismethylene (—CH₂—) and L is sulfonylmethylene (—SO₂—CH₂—); one X ismethylene (—CH₂—) and one X is carbonyl (—CO—) and L ismethylenescarbonyl (—CH₂—CO—); and in certain embodiments, one X iscarbonyl (—CO—) and one X is methylene (—CH₂—) and L iscarbonylmethylene (—CO—CH₂—), wherein R¹⁷ is selected from hydrogen,methyl, and ethyl.

In certain embodiments of a compound of Formula (1), a is 1, X and L arethe same, and each of X and L is selected from a bond (“-”), methylene(—CH₂—), hydroxymethylene (—C(OH)H—), ethane-1,1-diyl (—CHCH₃—),propane-2,2-diyl (—C(CH₃)₂—), oxygen (—O—), sulfonyl (—SO₂—), and amino(—NR¹⁷—), wherein R¹⁷ is selected from hydrogen and methyl.

In certain embodiments of a compound of Formula (1), a is 2; and L isselected from ethane-1,2-diyl (—CH₂—CH₂—), propane-1,2-diyl (—CH₂—CHCH₃—or —CHCH₃—CH₂—), hydroxyethane-1,2-diyl (—CH₂—CHOH—) or (—CHOH—CH₂—),carbonyl amino (—CO—NR¹⁷—), amino carbonyl (—NR¹⁷—CO—), methyleneamino(—CH₂—NR¹⁷—), aminomethylene (—NR¹⁷—CH₂—), methyleneoxy (—CH₂—O—),oxymethylene (—O—CH₂—), methylenethiyl (—CH₂—S—), thiylmethylene(—S—CH₂—), methylenesulfonyl (—CH₂—SO₂—), and sulfonylmethylene(—SO₂—CH₂—), wherein R¹⁷ is selected from hydrogen and methyl.

In certain embodiments of a compound of Formula (1),

at least one of R¹ and R⁵ is independently selected from, halogen,—N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO, —CN, —COOR¹⁰,—CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, substituted C₁₋₄ alkyl, C₁₋₄ alkoxy,substituted C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl,C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, C₃₋₅ cycloalkyloxy, and C₄₋₈cycloalkylalkyl;

each R¹⁰ is independently selected from hydrogen, deuterio, C₁₋₄ alkyl,and C₁₋₄ alkoxy, or two geminal R¹⁰ together with the nitrogen to whichthey are bonded form a 3- to 6-membered heterocyclic ring; and

one of R¹, R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

at least one of R¹ and R⁵ is independently selected from halogen,—N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy,C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;

each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to5-membered heterocyclic ring; and

one of R¹, R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

each of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH,C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl,C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, C₃₋₅ cycloalkyloxy, and C₄₋₈cycloalkylalkyl;

each R¹⁰ is independently selected from hydrogen, deuterio, C₁₋₄ alkyl,and C₁₋₄ alkoxy, or two geminal R¹⁰ together with the nitrogen to whichthey are bonded form a 3- to 6-membered heterocyclic ring; and

one of R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

each of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy,C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;

each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to5-membered heterocyclic ring; and

one of R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

one of R¹ and R⁵ is selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂,—N(R¹⁰)(OR¹⁰), —NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl,C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl,C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄fluoroalkoxy, C₃₋₅ cycloalkyl, C₃₋₅ cycloalkyloxy, and C₄₋₈cycloalkylalkyl;

each R¹⁰ is independently selected from hydrogen, deuterio, C₁₋₄ alkyl,and C₁₋₄ alkoxy, or two geminal R¹⁰ together with the nitrogen to whichthey are bonded form a 3- to 6-membered heterocyclic ring; and

one of R¹, R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

one of R¹ and R⁵ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂,—NO, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄alkylsulfinyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, andC₃₋₅ cycloalkyloxy;

each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to5-membered heterocyclic ring; and

one of R¹, R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

each of the other of R¹, R², R³, R⁴, and R⁵ is independently is selectedfrom hydrogen, deuterio, halogen, —N(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO,—OH, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl,C₁₋₄ alkylsulfonyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₁₋₄ alkyl,C₁₋₄ alkoxy, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, and C₄₋₈cycloalkylalkyl; and

each R¹⁰ is independently selected from hydrogen and C₁₋₄ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to6-membered heterocyclic ring.

In certain embodiments of a compound of Formula (1),

each of the other of R¹, R², R³, R⁴, and R⁵ is independently selectedfrom hydrogen, deuterio, halogen, —NR¹⁰2, —N(R¹⁰)(OR¹⁰), —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ fluoroalkyl, and C₁₋₄fluoroalkoxy, and

each R¹⁰ is independently selected from hydrogen and C₁₋₄ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to5-membered heterocyclic ring.

In certain embodiments of a compound of Formula (1), each of R¹ and R⁵is hydrogen.

In certain embodiments of a compound of Formula (1), each of the otherof R¹, R², R³, R⁴, and R⁵ is hydrogen.

In certain embodiments of a compound of Formula (1), each of R², R³, andR⁵ is hydrogen.

In certain embodiments of a compound of Formula (1), R1 is selected fromhalogen, —N(R10)2, —N+(—O—)(R¹⁰)₂, —N(OR¹⁰)(R¹⁰), —NO₂, —NO,—N(R¹⁰)(S(═O)R¹⁰), —N(R¹⁰)(S(═O)₂R¹⁰), —N(R¹⁰)(C(O)R¹⁰),—N(R¹⁰)(C(O)OR¹⁰), —N(R¹⁰)(C(O)N(R¹⁰)₂, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH,—SH, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl,—S(O)N(R¹⁰)₂, —S(O)₂N(R¹⁰)₂, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₁₋₆alkyl, substituted C₁₋₆ alkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy,C₃₋₆ cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyloxy,substituted C₃₋₆ cycloalkyloxy, C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂cycloalkylalkyl, C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, C₇₋₁₆ arylalkyl,substituted C₇₋₁₆ arylalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ heteroalkoxy, substituted C₁₋₆ heteroalkoxy, C₃₋₆heterocycloalkyl, substituted C₃₋₆ heterocycloalkyl, C₄₋₁₂heterocycloalkylalkyl, substituted C₄₋₁₂ heterocycloalkylalkyl, C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₆₋₁₆ heteroarylalkyl, andsubstituted C₆₋₁₆ heteroarylalkyl; wherein each R¹⁰ is independentlyselected from hydrogen, deuterio, C₁₋₄ alkyl, and C₁₋₄ alkoxy, or twogeminal R¹⁰ together with the nitrogen to which they are bonded form a3- to 6-membered heterocyclic ring; and

R⁵ is hydrogen.

In certain embodiments of a compound of Formula (1),

R¹ selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂,—NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ heteroalkyl,C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl,C₃₋₅ cycloalkyloxy, and C₄₋₈ cycloalkylalkyl; wherein each R¹⁰ isindependently selected from hydrogen, deuterio, C₁₋₄ alkyl, and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring; and

R⁵ is hydrogen.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl,or two R¹⁰ together with the nitrogen to which they are bonded form a 3-to 5-membered heterocyclic ring; and

R⁵ is hydrogen.

In certain embodiments of a compound of Formula (1),

each of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH,C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl,C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, C₃₋₅ cycloalkyloxy, and C₄₋₈cycloalkylalkyl; wherein each R¹⁰ is independently selected fromhydrogen, deuterio, C₁₋₄ alkyl, and C₁₋₄ alkoxy, or two geminal R¹⁰together with the nitrogen to which they are bonded form a 3- to6-membered heterocyclic ring;

one of R², R³, and R⁴ is —N(—CH₂—CH₂—R⁹)₂, —CH₂—N(—CH₂—CH₂—R⁹)₂,—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—O—N(—CH₂—CH₂—R⁹)₂, —CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂,—CH₂—CO—N(—CH₂—CH₂—R⁹)₂, —O—CO—N(—CH₂—CH₂—R⁹)₂, and—CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ is independently selectedfrom —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R², R³, and R⁴ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

each of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy,C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy; wherein each R¹⁰ isindependently selected from hydrogen and C₁₋₃ alkyl, or two R¹⁰ togetherwith the nitrogen to which they are bonded form a 3- to 5-memberedheterocyclic ring;

one of R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of the other R², R³, and R⁴ is hydrogen;

R⁶ is —COOH;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —O—, —NR¹⁷—,—CH₂—CH₂—), —CH₂—CHCH₃—, —CHCH₃—CH₂—, —CH₂—CHOH—, —CHOH—CH₂—, —CO—NR¹⁷⁻,—NR¹⁷—CO—, —CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—,—CH₂—SO₂—, and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰),—NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ heteroalkyl,C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl,C₃₋₅ cycloalkyloxy, and C₄₋₈ cycloalkylalkyl; wherein each R¹⁰ isindependently selected from hydrogen, deuterio, C₁₋₄ alkyl, and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring;

one of R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R², R³, R⁴, and R⁵ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl,or two R¹⁰ together with the nitrogen to which they are bonded form a 3-to 5-membered heterocyclic ring;

one of R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of the other of R², R³, R⁴, and R⁵ is hydrogen;

R⁶ is —COOH;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —O—, —NR¹⁷—,—CH₂—CH₂—), —CH₂—CHCH₃—, —CHCH₃—CH₂—, —CH₂—CHOH—, —CHOH—CH₂—, —CO—NR¹⁷—,—NR¹⁷—CO—, —CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—,—CH₂—SO₂—, and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1),

R⁵ is selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰),—NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ heteroalkyl,C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl,C₃₋₅ cycloalkyloxy, and C₄₋₈ cycloalkylalkyl; wherein each R¹⁰ isindependently selected from hydrogen, deuterio, C₁₋₄ alkyl, and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring;

one of R¹, R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R¹, R², R³, and R⁴ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

R⁵ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl,or two R¹⁰ together with the nitrogen to which they are bonded form a 3-to 5-membered heterocyclic ring;

one of R¹, R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of the other of R¹, R², R³, and R⁴ is hydrogen;

R⁶ is —COOH;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —O—, —NR¹⁷—,—CH₂—CH₂—), —CH₂—CHCH₃—, —CHCH₃—CH₂—, —CH₂—CHOH—, —CHOH—CH₂—, —CO—NR¹⁷—,—NR¹⁷—CO—, —CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—,—CH₂—SO₂—, and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1),

one of R¹ and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R¹, R², R³, R⁴, and R⁵ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

one of R¹ and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R¹, R², R³, R⁴, and R⁵ is hydrogen;

R⁶ is —COOH;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —O—, —NR¹⁷—,—CH₂—CH₂—), —CH₂—CHCH₃—, —CHCH₃—CH₂—, —CH₂—CHOH—, —CHOH—CH₂—, —CO—NR¹⁷⁻,—NR¹⁷—CO—, —CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—,—CH₂—SO₂—, and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰),—NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ heteroalkyl,C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl,C₃₋₅ cycloalkyloxy, and C₄₋₈ cycloalkylalkyl; wherein each R¹⁰ isindependently selected from hydrogen, deuterio, C₁₋₄ alkyl, and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring;

R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂, —CH₂—N(—CH₂—CH₂—R⁹)₂,—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—O—N(—CH₂—CH₂—R⁹)₂, —CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂,—CH₂—CO—N(—CH₂—CH₂—R⁹)₂, —O—CO—N(—CH₂—CH₂—R⁹)₂, and—CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ is independently selectedfrom —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of R², R³, and R⁵ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl;or two R¹⁰ together with the nitrogen to which they are bonded to form a3- to 5-membered heterocyclic ring;

R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂, —CH₂—N(—CH₂—CH₂—R⁹)₂,—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—O—N(—CH₂—CH₂—R⁹)₂, —CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂,—CH₂—CO—N(—CH₂—CH₂—R⁹)₂, —O—CO—N(—CH₂—CH₂—R⁹)₂, and—CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ is independently selectedfrom —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of R², R³, and R⁵ is hydrogen;

R⁶ is —COOH;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy;

L is selected from a bond “-”, —CH₂—, —C(OH)H—, —CHCH₃—, —O—, —NR¹⁷—,—CH₂—CH₂—), —CH₂—CHCH₃—, —CHCH₃—CH₂—, —CH₂—CHOH—, —CHOH—CH₂—, —CO—NR¹⁷—,—NR¹⁷—CO—, —CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—,—CH₂—SO₂—, and —SO₂—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the beta-carbon atom is (R).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the beta-carbon atom is (S).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the β carbon atom is of the (R) configuration, theabsolute axial stereochemistry (atropisomerism) is R_(a), and theabsolute stereochemistry of a compound of Formula (1) is (R,R_(a)).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the 3 carbon atom is of the (R) configuration, theabsolute axial stereochemistry (atropisomerism) is S_(a), and theabsolute stereochemistry of a compound of Formula (1) is (R,S_(a)).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the β carbon atom is of the (S) configuration, theabsolute axial stereochemistry (atropisomerism) is R_(a), and theabsolute stereochemistry of a compound of Formula (1) is (S,R_(a)).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the 3 carbon atom is of the (S) configuration, theabsolute axial stereochemistry (atropisomerism) is S_(a), and theabsolute stereochemistry of a compound of Formula (1) is (S,S_(a)).

In certain embodiments, a compound of Formula (1) is selected from:

-   4-Amino-3-[3-[bis(2-chloroethyl)amino]phenyl]butanoic acid (1);-   4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid    (2);-   4-Amino-3-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]butanoic    acid (3);-   4-Amino-3-[3-[bis(2-chloroethyl)amino]phenyl]-3-methyl-butanoic acid    (4);-   4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoic    acid (5);-   4-Amino-3-[3-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoic    acid (6);-   4-Amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoic    acid (7);-   4-Amino-3-[5-[bis(2-chloroethyl)carbamoyl]-2-methyl-phenyl]butanoic    acid (8);-   4-Amino-3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoic    acid (9);-   4-Amino-3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoic acid    (10);-   4-Amino-3-[5-(bis(2-iodoethyl)amino)-2-methyl-phenyl]butanoic acid    (11);-   4-Amino-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoic    acid (12);-   4-Amino-3-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]butanoic    acid (13);-   4-Amino-3-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoic    acid (14);-   4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid    (15);-   4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-2-fluoro-butanoic    acid (16);-   3-(Aminomethyl)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-hydroxy-butanoic    acid (17);-   4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-nitro-phenyl]butanoic acid    (18);-   [3-Amino-2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinic    acid (19);-   3-[1-(Aminomethyl)-2-(1H-tetrazol-5-yl)ethyl]-N,N-bis(2-chloroethyl)-4-methyl-aniline    (20);-   4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]butanoic    acid (21);-   4-Amino-3-[5-[bis(2-chloroethyl)aminomethyl]-2-methyl-phenyl]butanoic    acid (22);-   4-Amino-3-[5-[bis(2-chloroethyl)carbamoyloxy]-2-methyl-phenyl]butanoic    acid (23);-   4-Amino-3-[4-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]-3-methyl-butanoic    acid (24); and-   4-[1-(Aminomethyl)-3-hydroxy-1-methyl-3-oxo-propyl]-N,N-bis(2-chloroethyl)-3-methyl-benzeneamine    oxide (25);-   or a pharmaceutically acceptable salt of any of the foregoing.

In certain embodiments of any of the foregoing compounds, apharmaceutically acceptable salt is the hydrochloride salt.

In certain embodiments of any of the foregoing compounds, apharmaceutically acceptable salt is the dihydrochloride salt.

In certain embodiments of a compound of Formula (1), a pharmaceuticallyacceptable salt is the hydrochloride salt.

In certain embodiments of a compound of Formula (1), a pharmaceuticallyacceptable salt is the dihydrochloride salt.

In certain embodiments, compounds of Formula (1) are selectivesubstrates for the LAT1/4F2hc transporter.

In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent V_(max) of at least 10% the V_(max) ofgabapentin. In certain embodiments, compounds provided by the presentdisclosure exhibit a LAT1/4F2hc-dependent V_(max) of at least 20% theV_(max) of gabapentin. In certain embodiments, compounds provided by thepresent disclosure exhibit a LAT1/4F2hc-dependent V_(max) of at least30% the V_(max) of gabapentin. In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependentV_(max) of at least 40% the V_(max) of gabapentin. In certainembodiments, compounds provided by the present disclosure exhibit aLAT1/4F2hc-dependent V_(max) of at least 50% the V_(max) of gabapentin.In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent V_(max) of at least 60% the V_(max) ofgabapentin. In certain embodiments, compounds provided by the presentdisclosure exhibit a LAT1/4F2hc-dependent V_(max) of at least 70% theV_(max) of gabapentin. In certain embodiments, compounds provided by thepresent disclosure exhibit a LAT1/4F2hc-dependent V_(max) of at least80% the V_(max) of gabapentin. In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependentV_(max) of at least 90% the V_(max) of gabapentin. In certainembodiments, compounds provided by the present disclosure exhibit aLAT1/4F2hc-dependent V_(max) of at least 100% the V_(max) of gabapentin.

In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L) and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 50% that of L-leucine measured at an extracellularconcentration of 1 mM (1 mmol/L). In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependent uptakeof at least 10% that of gabapentin measured at an extracellularconcentration of 1 mM (1 mmol/L); and a system A-, system N-, a systemASC-, and a LAT2/4F2hc-dependent uptake of less than 40% that ofL-leucine measured at an extracellular concentration of 1 mM (1 mmol/L).In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L); and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 30% that of L-leucine measured at anextracellularconcentration of 1 mM (mmol/L). In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependent uptakeof at least 10% that of gabapentin measured at an extracellularconcentration of 1 mM (1 mmol/L); and a system A-, system N-, a systemASC-, and a LAT2/4F2hc-dependent uptake of less than 20% that ofL-leucine measured at an extracellular concentration of 1 mM (1 mmol/L).In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L); and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 10% that of L-leucine measured at an extracellularconcentration of 1 mM (1 mmol/L). In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependent uptakeof at least 10% that of gabapentin measured at an extracellularconcentration of 1 mM (1 mmol/L); and a system A-, system N-, a systemASC-, and a LAT2/4F2hc-dependent uptake of less than 5% that ofL-leucine measured at an extracellular concentration of 1 mM (1 mmol/L).In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L); and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 1% that of L-leucine measured at an extracellularconcentration of 1 mM (1 mmol/L).

Compounds of Formula (1) may be adapted as prodrugs to achieve desirablepharmacokinetic properties. For example, suitable prodrugs ofβ-substituted γ-amino acid derivatives and β-substituted γ-amino acidanalogs are disclosed by Gallop, et al., U.S. Pat. No. 7,109,239, U.S.Pat. No. 6,972,341, U.S. Pat. No. 6,818,787 and U.S. Pat. No. 7,227,028.Prodrugs of compounds of Formula (1) include the prodrug systemsdisclosed by Gallop, et al., as well as others known in the art.

Synthesis of Compounds

Compounds disclosed herein may be obtained via the general syntheticmethods illustrated in Schemes 1-10. General synthetic methods useful inthe synthesis of compounds, precursors, and starting materials describedherein are available in the art. Starting materials useful for preparingcompounds and intermediates thereof, and/or for practicing methodsdescribed herein, are commercially available or may be prepared bywell-known synthetic methods (March's Advanced Organic Chemistry:Reactions, Mechanisms, M. B. Smith, 7^(th) Edition, John Wiley & Sons,Hoboken, N.J., USA, 2013; Advanced Organic Chemistry: Part B: Reactionand Synthesis, Carey and Sundberg, 5^(th) Edition, Springer, Germany,2010; Comprehensive Organic Transformations, 2^(nd) Edition, Larock,Wiley-VCH, Weinheim, Germany, 1999; Science of Synthesis: Houben-WeylMethods of Moleculer Transformations, Thieme, Germany (www.thieme.de).

Additionally, as will be apparent to those skilled in the art, use ofconventional protecting groups or protecting strategies may be necessaryto prevent certain functional groups from undergoing undesiredreactions. Suitable protecting groups for various functional groups aswell as suitable conditions for protecting and deprotecting particularfunctional groups are well known in the art (Wuts and Greene, Greene'sProtective Groups in Organic Synthesis, ^(4th) Ed, 2007,Wiley-Interscience, John Wiley & Sons, Inc., Hoboken, N.J.).

It will be appreciated that where typical or preferred processconditions, e.g., reaction temperatures, reaction times, molar ratios ofreactants, solvents, pressures, etc., are given other process conditionsmay also be used. Optimal reaction conditions may vary with theparticular reactants, solvents, functional groups, and protecting groupsused, but such conditions may be determined by one skilled in the art byroutine optimization procedures.

Furthermore, certain compounds provided by the present disclosure maycontain one or more stereogenic centers. Accordingly, and if desired,such compounds may be prepared or isolated as pure stereoisomers, e.g.,as individual enantiomers, diastereomers, atropisomers, rotamers, or asstereoisomer enriched mixtures or racemates. All such stereoisomers areincluded within the scope of this disclosure. Pure stereoisomers (orenriched mixtures thereof) may be prepared using, for example, opticallyactive starting materials, stereoselective reagents such as chiralcatalysts and auxiliaries well known in the art. Alternatively, racemicmixtures of such compounds may be separated or partially enriched using,for example, chromatographic methods with chiral stationary phases,chiral resolving agents, and the like, also well known in the art andeasily adaptable to the particular compound to be separated.

Methods of preparing (protected) 3-, 4-, and/or 5-ring-substituted andring-unsubstituted pyrrolidin-2-ones (γ-lactams) from commercial orknown starting materials are well known in the art (Huang, in NewMethods for the Asymmetric Synthesis of Nitrogen Heterocycles; ResearchSignpost: Trivandrum, India, 2005, pp 197-222; Smith, in Science ofSynthesis (S. Weinreb, et. al (Ed.s)), Georg Thieme Verlag: Stuttgart,Germany, 2005, Vol. 21, pp 647-711). In particular, methods of preparing(protected) 4-aryl-, 4-alkylaryl-, 4-heteroalkylaryl, 4-aryloxysubstituted pyrrolidin-2-ones from commercial or known startingmaterials are well known in the art. In certain embodiments, 3-, 4-,and/or 5-ring substituted and unsubstituted pyrrolidin-2-ones may beused as starting materials for the preparation of N-mustardβ-substituted γ-amino acid derivatives and β-substituted γ-amino acidanalogs or (bio)isosteres provided by the present disclosure.

Methods of synthetic manipulations and modifications of the underlyingpyrrolidin-2-one ring (γ-lactam ring) of 3-, 4-, and/or5-ring-substituted and -unsubstituted pyrrolidin-2-ones are also wellknown in the art (Huang, in New Methods for the Asymmetric Synthesis ofNitrogen Heterocycles; Research Signpost: Trivandrum, India, 2005, pp197-222; Smith, in Science of Synthesis (S. Weinreb, et. al (Ed.s)),Georg Thieme Verlag: Stuttgart, Germany, 2005, Vol. 21, pp 647-711). Incertain embodiments, the underlying pyrrolidin-2-one ring (γ-lactam) maybe modified to allow for regio- and/or stereoselective incorporation ofauxiliary molecular functionalities into the resulting β-substitutedγ-amino acid scaffold of the final N-mustard functionalizedβ-substituted γ-amino acid derivatives or β-substituted γ-amino acidanalogs or (bio)isosteres. Auxiliary molecular functionalities may, forexample, be incorporated to modulate interaction with LAT1 transporterproteins, e.g., efficacy of translocation through biological membranes,modulate physiochemical or ADMET properties, or to modulate the activityof the physiologically active N-mustard moiety, e.g., cytotoxicity.

Methods of synthetic manipulations and modifications of the aryl ring of(protected) 4-aryl-, 4-alkylaryl-, 4-heteroalkylaryl, 4-aryloxysubstituted pyrrolidin-2-ones are also well known in the art (Xu, etal., U.S. Pat. No. 8,344,028 (2013)). In certain embodiments, theunderlying aryl ring may be modified to allow for regioselectiveincorporation of functional groups that can be converted to N-mustardsby using reagents, methods, and protocols well known in the art. Incertain embodiments, the underlying aryl ring may also be modified toallow for regio- and/or stereoselective incorporation of auxiliarymolecular functionalities into the arene scaffold. Auxiliary molecularfunctionalities may, for example, be incorporated to modulateinteraction with LAT1 transporter proteins, e.g., efficacy oftranslocation through biological membranes (binding to theLAT1-transporter protein and capacity of LAT1-mediated transport),modulate physiochemical or ADMET properties, or to modulate the activityof the physiologically active N-mustard moiety, e.g., cytotoxicity.

Methods of functionalizing the amino acid scaffold of 3-aryl-,3-alkylaryl-, 3-heteroalkylaryl, 3-aryloxy substituted 2-, 3-, and/or4-substituted and unsubstituted γ-amino acid amino acid derivatives(β-substituted γ-amino acid butyric acid derivatives, β-substituted GABAderivatives), similarly substituted 3-aminopropylphosphinic acid,3-aminopropylsulfinic acids, or 3-aminopropyl substituted carboxylicacid (bio)isosteres from commercial or known starting materials are alsowell known in the art.

In certain embodiments, starting materials may be used in their fullyprotected form wherein the amino group or a synthetic equivalent or aprecursor thereof and the carboxylic acid, phosphinic acid, sulfinicacid, carboxylic acid (bio)isosteres or synthetic equivalents orprecursors of any of the foregoing are appropriately protected.

In certain embodiments, starting materials may be used in theirhemi-protected form wherein the amino group or a synthetic equivalent ora precursor thereof is protected and the carboxylic acid group,phosphinic acid, sulfinic acid, or a carboxylic acid (bio)isosterefunctional group or synthetic equivalents or precursors of any of theforegoing are unprotected or free.

In certain embodiments, starting materials may be used in theirhemi-protected form wherein the amino group is unprotected or free andthe carboxylic acid, phosphinic acid, sulfinic acid, or a carboxylicacid (bio)isostere or synthetic equivalents or precursors of any of theforegoing are appropriately protected.

In certain embodiments, starting materials may be used in their fullyunprotected form wherein the amino group and the carboxylic acid,phosphinic acid, sulfinic acid, or the carboxylic acid (bio)isostere orsynthetic equivalents or precursors of any of the foregoing areunprotected.

Methods for converting 4-aryl-, 4-alkylaryl-, 4-heteroalkylaryl,4-aryloxy substituted pyrrolidin-2-ones pyrrolidin-2-ones to theircorresponding ring-opened derivatives such as 3-aryl-, 3-alkylaryl-,3-heteroalkylaryl, 3-aryloxy substituted 4-aminobutyric acid derivativesare known in the art (Ordfiez and Cativiela, Tetrahedron: Asymmetry,2007, 18, 3-99; M. Ordfiez, et al., Tetrahedron: Asymmetry, 2010, 21,129-147; and Xu, et al., U.S. Pat. No. 8,344,028). Such ring-openedderivatives may be prepared either (a) in their fully unprotected form(e.g., with a free amino group and a free carboxylic acid group,zwitterions thereof, any salts thereof, or any pharmaceuticallyacceptable salts thereof), (b)(i) in their amino-protected form with afree carboxylic acid group, any salts thereof, or any pharmaceuticallyacceptable salts thereof), (b)(ii) in their carboxyl-protected form anda free amino group, any salts thereof including pharmaceuticallyacceptable salts, or synthetic equivalents or precursors thereof), or(c) in their fully amino- and carboxylic acid-protected form orsynthetic equivalents or precursors thereof. Alternatively, methods ofconverting any of the ring-opened fully protected, hemi-protected, orunprotected 3-aryl-, 3-alkylaryl-, 3-heteroalkylaryl, 3-aryloxysubstituted 4-aminobutyric acid derivatives, to the corresponding4-aryl-, 4-alkylaryl-, 4-heteroalkylaryl, 4-aryloxy substitutedpyrrolidin-2-ones are also well known in the art.

Many other methods for the preparation of appropriately functionalizedor substituted 3-aryl-, 3-alkylaryl-, 3-heteroalkylaryl, 3-aryloxysubstituted (β-aryl-, β-alkylaryl-, β-heteroalkylaryl, β-aryloxysubstituted) 4-aminobutyric acid analogs, carboxylic acid(bio)isosteres, derivatives, or precursors thereof using commercial orknown starting materials described herein are either described in theart or will be readily apparent to one skilled in the art in view of thereferences provided herein. Accordingly, the methods presented in theschemes provided by the present disclosure are illustrative rather thancomprehensive.

Referring to Scheme 1, selected and representative starting materialsfor the preparation N-mustard functionalized 4-aryl-, 4-alkylaryl-,4-heteroalkylaryl, 4-aryloxy substituted pyrrolidin-2-ones, or 3-aryl-,3-alkylaryl-, 3-heteroalkylaryl, 3-aryloxy substituted (β-aryl-,β-alkylaryl-, β-heteroalkylaryl, β-aryloxy substituted) γ-amino acidanalogs or carboxylic acid (bio)isosteres are compounds of Formula (A)and Formula (B).

Referring to Scheme 1, in certain embodiments R¹ and/or R⁵, and thelinker L are as defined herein; one of R², R³, and R⁴ in compounds ofFormula (A) and Formula (B) is -E-MH, wherein E is a bond (“-”), anoxygen atom (—O—), a methylene group (—CH₂—), a methyleneoxy group(—CH₂—O—), a carbonyl group (—CO—), or a methylenecarbonyl group(—CH₂—CO—), and wherein -MH is an amino group (—NH₂), a hydroxyl group(—OH), or a sulfhydryl group (—SH). Each of the other remaining R², R³,and R⁴ is hydrogen; each R⁷; and each R⁸ is hydrogen.

Referring to Scheme 1, for example (a) -E-MH is equivalent to a primaryaromatic amino group (—NH₂, aniline) when E is a bond (“-”) and MH is anamino group (—NH₂), (b) -E-MH is equivalent to a primary O-arylhydroxylamino group (—O—NH₂) when E is an oxygen atom (—O—) and MH is anamino group (—NH₂), (c) -E-MH is equivalent to a primary aminomethylgroup (—CH₂—NH₂, primary benzylic amine) when E is a methylene group(—CH₂—) and MH is an amino group (—NH₂), (d) -E-MH is equivalent to anaromatic hydroxyl group (—OH, phenol) when E is a bond (“-”) and MH is ahydroxyl group (—OH), (e) -E-MH is equivalent to a hydroxymethyl group(—CH₂—OH, benzylic alcohol) when E is a methylene group (—CH₂—) and MHis a hydroxyl group (—OH), (f) -E-MH is equivalent to a primaryO-benzylic hydroxylamino group (—CH₂—O—NH₂) when E is a methyleneoxygroup (—CH₂—O—) and MH is an amino group (—NH₂), (g) -E-MH is equivalentto an aromatic sulhydryl group (—SH, thiophenol derivative) when E is abond (“-”) and MH is a hydroxyl group (—OH), (h) -E-MH is equivalent toa methylenesulhydryl group (—CH₂—SH, benzylic thiol) when E is amethylene group (—CH₂—) and MH is a sulfhydryl group (—SH), (i) -E-MH isequivalent to an aromatic carboxylic acid group (—CO—OH, benzoic acid)when E is a carbonyl group (—C(═O)—) and MH is a hydroxyl group (—OH),(j) -E-MH is equivalent to a carboxylic acid group (—CO—OH, benzoicacid) when E is a methylenecarbonyl group (—CH₂—C(═O)—) and MH is ahydroxyl group (—OH).

It will be obvious to the one skilled in the art that in someembodiments of the disclosure the group “-E-” in functional groups -E-MHpresented in the following schemes is equivalent to the group -A- in thedefinition of the composition of a chemotherapeutic moiety as describedherein.

The linker L is as defined herein.

Referring to Scheme 1, in certain embodiments R²⁰ in compounds ofFormula (B) is a protected carboxyl group such as a lower alkyl ester ofa carboxyl group, e.g., a methyl, ethyl, or tert-butyl ester, or abenzyl ester derivative, e.g., benzyl, pentamethylbenzyl or(4-methoxy)benzyl. In certain embodiments, R²⁰ in compounds of Formula(B) is a tert-butyl ester group (CO₂tBu). In certain embodiments, R²⁰ incompounds of Formula (B) is a methyl ester group (CO₂Me).

Referring to Scheme 1, in certain embodiments R²⁰ in compounds Formula(B) is a protected phosphinic acid derivative, e.g.,1,1-diethyloxyethylethoxyphosphino-1-one (—P(═O)(OEt)[C(OEt)₂Me] (U.S.Pat. No. 8,344,028; Baylis, Tetrahedron Letter, 1995, 36(51), 9385-9388;and Burgos-Lepley, et al., Bioorg. Med. Chem. Lett., 2006, 16,2333-2336).

Referring to Scheme 1, in certain embodiments R²⁰ in compounds Formula(B) is a protected sulfinic acid precursor derivative, e.g., a2-mercaptobenzothiazole (Carruthers, et al., Bioorg. Med. Chem. Lett,1995, 5, 237-240; Carruthers, et al., Bioorg. Med. Chem. Lett, 1998, 5,3059-3064; Okawara, et al., Chem. Lett., 1984, 2015; and Burgos-Lepley,et al., Bioorg. Med. Chem. Lett., 2006, 16, 2333-2336).

Referring to Scheme 1, in certain embodiments R²⁰ in compounds Formula(B) is a unprotected or protected carboxylic acid (bio)isostereincluding a protected or unprotected 1H-tetrazole (Ballatore, et al.,ChemMedChem, 2013, 8(3), 385-395; Bryans, et al., U.S. Pat. No.6,518,289; Burgos-Lepley, et al., Bioorg. Med. Chem. Lett., 2006, 16,2333-2336).

Referring to Scheme 1, in certain embodiments of compounds of Formula(A) Q is NH or N-PG where PG is a suitable nitrogen protecting group,e.g., tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz, Z),(R/S)-1-phenyl-ethoxycarbonyl, (R)-1-phenyl-ethoxycarbonyl,(S)-1-phenyl-ethoxycarbonyl, 1-methyl-1-phenyl-ethoxycarbonyl,triphenylmethyl (trityl), or the like. In certain embodiments ofcompounds of Formula (A) Q is NH (non-protected lactam). In certainembodiments of compounds of Formula (A) and of Formula (C), Q is N-Boc(NCO₂tBu) (N-protected lactam).

Referring to Scheme 1, in certain embodiments of compounds of Formula(B) Q is N(H)—PG where PG is a suitable nitrogen protecting group, e.g.,tert-butoxycarbonyl (Boc), allyloxycarbonyl (alloc), benzyloxycarbonyl(Cbz, Z), ethoxycarbonyl, methoxycarbonyl,(R/S)-1-phenyl-ethoxycarbonyl, (R)-1-phenyl-ethoxycarbonyl,(S)-1-phenyl-ethoxycarbonyl, 1-methyl-1-phenyl-ethoxycarbonyl, formyl,acetyl, trifluoroacetyl, benzoyl, triphenylmethyl (trityl),4-methoxyphenyl-diphenylmethyl, di-(4-methoxyphenyl)-phenylmethyl, orthe like. In certain embodiments, PG in compounds of Formula (B) istert-butoxycarbonyl (Boc) and Q is N(H)Boc (N(H)CO₂tBu). In certainembodiments of compounds of Formula (B) PG is acetyl and Q isN(H)—Ac(N(H)COMe). In certain embodiments of compounds of Formula (B)and of Formula (D), PG is benzoyl and Q is N(H)—Bz (N(H)COPh).

Referring to Scheme 1, in certain embodiments Q is N(PG)₂, where PG is anitrogen protecting group such as an imide-type protecting group, e.g.,phthalyl or tert-butoxycarbonyl (Boc). In certain embodiments ofcompounds of Formula (B) PG is phthalyl and Q is N(phthalyl). In certainembodiments of compounds of Formula (B) PG is tert-butoxycarbonyl and Qis N(Boc)₂.

Referring to Scheme 1, in certain embodiments the protected aminefunctionality is an imine where Q is N═CR³⁰R³¹ and each of R³⁰ and R³¹is independently selected from branched C₁₋₄ alkyl, non-branched C₁₋₄alkyl, substituted aryl, non-substituted aryl, substituted heteroaryl,and non-substituted heteroaryl.

The structures presented in the schemes provided by the presentdisclosure are illustrative rather than comprehensive.

Referring to Scheme 2, in certain embodiments R¹ and/or R⁵, R²⁰, E, thelinker L, and the protecting groups PG and Q are as defined herein; oneof R², R³, and R⁴ in compounds of Formula (C) and Formula (D) is -E-NH₂,wherein E is a bond (“-”), an oxygen atom (—O—), a methylene group(—CH₂—), or methylenoxy group (—CH₂—O—), and MH is an amino group (—NH₂)so that -E-NH₂ is equivalent to (a) a primary aromatic amino group(—NH₂, aniline), (b) a primary O-aryl hydroxylamino group (—O—NH₂), (c)a primary aminomethyl group (—CH₂—NH₂), or (d) a primary O-benzylhydroxylamino group (—CH₂—O—NH₂). Each of the other remaining R², R³,and R⁴ is hydrogen; and each R⁷ and each R⁸ is hydrogen. X is a suitableleaving group e.g., chloro (—Cl) or bromo (—Br).

Referring to Scheme 2, conversion of the primary amino group as incompounds of Formula (C) and of Formula (D) to theN,N-bis-(2-hydroxyethyl) amino group (N,N-bis-(2-hydroxyethylation)) asin compounds of Formula (E) or of Formula (F) may be accomplished byreacting compounds of Formula (C) and of Formula (D) in suitablesolvents such as about 25-75 vol.-% aqueous acetic acid (HOAc), glacialacetic acid, water, tetrahydrofuran (THF), ethanol (EtOH), 1,4-dioxane,or mixtures of any of the foregoing with an excess of ethylene oxide(oxirane) (about 4-20 equivalents) at a temperature of about −20° C. toabout room temperature for about 12-48 hours. Alternatively, thereaction mixture may be heated in a sealed reaction vessel from about80-140° C. for comparable times (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Jordan, et al., Bioorg. Med. Chem., 2002, 10(8),2625-2633; Abela Medici, et al, J. Chem. Soc., Perkin Trans. 1, 1997,(20), 2258-2263; Feau, et al., Org. Biomolecular Chem., 2009, 7(24),5259-5270; Springer, et al., J. Med. Chem., 1990, 33(2), 677-681;Taylor, et al., Chem. Biol. Drug Des., 2007, 70(3), 216-226; Buss, etal., J. Fluorine Chem., 1986, 34(1), 83-114; Larden and Cheung,Tetrahedron Lett., 1996, 37(42), 7581-7582; Spreitzer and Puschmann,Monatshefte fiir Chemie, 2007, 138(5), 517-522; Niculesscu-Duvaz, etal., J. Med. Chem., 2004, 47(10), 2651-2658; Weisz, et al., Bioorg. Med.Chem. Lett., 1995, 5(24), 2985-2988; Thorn, et al., J. Org. Chem, 1975,40(11), 1556-1558; Baraldini, et al., J. Med., Chem., 2000, 53(14),2675-2684; Zheng, et al., Bioorg., Med., Chem., 2010, 18(2), 880-886;Gourdi, et al., J., Med., Chem., 1990, 33(4), 1177-1186; Haines, et al.,J. Med. Chem., 1987, 30, 542-547; Matharu, et al., Bioorg. Med. Chem.Lett., 2010, 20, 3688-3691; and Kupczyk-Subotkowska, et al., J. DrugTargeting, 1997, 4(6), 359-370).

Referring to Scheme 2, conversion of the primary amino group as incompounds of Formula (C) and of Formula (D) to theN,N-bis-(2-hydroxyethyl) amino group (N,N-bis-(2-hydroxyethylation)) asin compounds of Formula (E) or of Formula (F) may be accomplished byreacting compounds of Formula (C) and of Formula (D) in suitablesolvents such water with an excess of about 2-5 equivalents of asuitable 2-halogeno ethanol derivative, e.g., 2-chloroethanol(ClCH₂CH₂OH) or 2-bromoethanol (BrCH₂CH₂OH), and about 2.0 equivalentsof a suitable inorganic base such as sodium bicarbonate (NaHCO₃), sodiumcarbonate (Na₂CO₃), or calcium carbonate (CaCO₃) at about refluxtemperature for about 8-24 hours. Optionally, the reaction may becarried out in the presence of a catalytic amount (about 10 mol-%) ofpotassium iodide (KI) (Palmer, et al., J. Med. Chem. 1990, 33(1),112-121; Coggiola, et al., Bioorg. Med. Chem. Lett., 2005, 15(15),3551-3554; Verny and Nicolas, J. Label. Cmpds Radiopharm., 1988, 25(9),949-955; and Lin, Bioorg. Med. Chem. Lett., 2011, 21(3), 940-943).

Referring to Scheme 3, in certain embodiments electron-deficient arylhalides of Formula (G) or Formula (H), activated with strongly electronwithdrawing substituents for nucleophilic aromatic substitutionreactions (SNAr) at the aryl ring, may be useful starting materials forincorporating N,N-bis-(2-functionalized) ethyl amino groups as incompounds of Formula (I) and Formula (J) where the correspondingN,N-bis-(2-functionalized)ethyl amino groups areN,N-bis-(2-hydroxyethyl) amino groups. Commonly used leaving groups (—X)for SNAr-reactions include halogeno, e.g., fluoro (—F), chloro (—Cl),bromo (—Br), with accessory activating groups at the 2- or 4-positionrelative to the leaving group (ortho- or para-positions). Such groupsdecrease the electron density in the arene ring and increase thesusceptibility to nucleophilic attack and displacement of the leavinggroup (—X). Examples of activating, strongly electron-withdrawing groups(EWG), include trifluoromethyl (—CF₃), cyano (—CN), nitro (—NO₂), amide(—CON(R¹⁰)₂), and formyl (—CHO).

Useful secondary amines for the introduction of theN,N-bis-(2-hydroxyethyl) amino functionality include diethanolamine(HN(CH₂CH₂OH)₂), protected diethanolamine derivatives, e.g.,O-benzylether protected diethanolamine (HN(CH₂CH₂OBn)₂), or precursorsof the putative N,N-bis-(2-hydroxyethyl)amino group, e.g., 3-pyrroline.Employing O-benzylether protected diethanolamine (HN(CH₂CH₂OBn)₂) or3-pyrroline necessitates conversion of the corresponding intermediatesubstitution products to compounds of Formula (I) and of Formula (J)bearing the target N,N-bis-(2-hydroxyethyl)amino groups using methodswell known in the art.

Referring to Scheme 3, in certain embodiments R¹ and/or R⁵, R¹⁰, R²⁰,the linker L, the protecting group PG, and Q, the electron withdrawinggroup (EWG), the leaving group (—X), and the secondary amine HNR₂ aredefined as described herein; R¹ and/or R⁵ may also represent an electronwithdrawing group (EWG); one or more of R², R³, and R⁴ in compounds ofFormula (G) or of Formula (H) is a suitable leaving group (—X)), one ormore of R², R³, and R⁴ is a electron withdrawing group (EWG) preferablyin 2- or 4-postion relative to the leaving group X; each of the otherremaining R², R³, and R⁴ is hydrogen; and each of R⁷ and R⁸ is hydrogen.

Referring to Scheme 3, N,N-bis(2-hydroxyethyl)amino derivatives as incompounds of Formula (I) or of Formula (J) may be prepared throughnucleophilic aromatic substitution reactions (SNAr) of aromatic halidesof Formula (G) and of Formula (H), activated by electron withdrawinggroups (EWGs), by reaction with an excess of about 1.5-5 equivalents ofthe neat amine, e.g., HN(CH₂CH₂OH)₂, HN(CH₂CH₂OBn)₂, or 3-pyrroline,(weakly basic reaction conditions) or solutions of the secondary aminein polar aprotic anhydrous solvents, e.g., anhydrous dimethylsulfoxide(DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc),acetonitrile (MeCN), 1,4-dioxane, tetrahydrofuran (THF), or mixtures ofthe foregoing at a temperature from about 80-200° C. (sealed tube), forabout 1-12 hours to provide N,N-bis(2-hydroxyethyl)amino-functionalizedcompounds of Formula (I) or of Formula (J). The reaction may also becarried out in the presence of a catalyst, e.g., copper powder (about 10mol-%) (Atwell, et al., J. Med. Chem., 2007, 50(6), 1197-1212; Palmer,et al., J. Med. Chem., 1994, 37, 2175-2184; Palmer, et al., J. Med.Chem., 1992, 35(17), 3214-3222; Palmer, et al., J. Med. Chem, 1990,33(1), 112-121; Davies, et al., J. Med. Chem. 2005, 48(16), 5321-5328;Jordan, et al., Bioorg. Med. Chem., 2002, 10(8), 2625-2633; Dheyongera,et al., Bioorg. Med. Chem., 2005, 13(3), 689-698; Lin, et al., Bioorg.Med. Chem. Lett., 2011, 21(3), 940-943; and Ferlin, et al., Bioorg. Med.Chem., 2004, 12(4), 771-777).

Referring to Scheme 3, methods to convert theN,N-bis-(2-benzyloxyethyl)amino group to a N,N-bis-(2-hydroxyethyl)aminogroup include, for example, catalytic hydrogenolysis of the benzyl ethergroups using heterogeneous catalysts, e.g., 5-10% Pd on carbon (Pd/C) orRaney®-Nickel under standard hydrogenation reaction conditions known inthe art (Vincent and Prunet, Tetrahedron Lett, 2006, 47(24), 4075-4077).

Referring to Scheme 3, conversion the 3-pyrroline ring of theN-aryl-3-pyrroline moiety to a N,N-bis-(2-hydroxyethyl)amino group as incompounds of Formula (G) or of Formula (H) include oxidative cleavage ofthe C═C-double with the Lemieux-Johnson reagent (osmium tetroxide/sodiumperiodate, OsO₄/NaIO₄) or by ozonolysis with an O₃/O₂-gas mixture.Reductive work-up, e.g., with borane-dimethylsulfide complex (BH₃.Me₂S),triphenylphosphine (Ph₃P), thiourea (C(═S)(NH₂)₂), or zinc dust, mayyield intermediate N,N-bis(2-oxoethyl)amino groups which maysubsequently be reduced to the desired N,N-bis-(2-hydroxyethyl)aminogroup as in compounds of Formula (G) or of Formula (H) with suitablereducing reagents, e.g., borane-THF complex (BH₃.THF), or sodiumborohydride (NaBH₄), under standard reaction conditions (Palmer andDenny, Synth. Commun., 1987, 17(5), 601-610).

In general, the biological activity of nitrogen mustards is based uponthe presence of a N,N-bis(2-chloroethyl) functionality. Thechemotherapeutic and cytotoxic effects are directly associated with thealkylation of DNA due to the strong electrophilic character of theN,N-bis(2-chloroethyl) functionality. Formation of covalent linkagesincluding interstrand crosslinks (ICLs) is highly cytotoxic and involvesthe disruption of fundamental cellular processes including DNAreplication leading to cellular death.

Many methods and reagents for converting primary alcohols to primaryalkyl chlorides including conversion of N,N-bis(2-hydroxyethyl)aminogroups to N,N-bis(2-chloroethyl)amino groups are known in the art. Themost common methods include the use of concentrated hydrochloric acid(HCl) and various inorganic chlorides of sulfur or phosphorus which areused either in neat form or as solutions in inert solvents such aschlorinated hydrocarbons, aromatic hydrocarbons, or polar non-proticsolvents, at room temperature or at elevated temperatures. Other usefulchlorination methods and reagents include, for example, combinations oftriphenyl phosphine and trichloroacetonitrile (Ph₃P/Cl₃CCN),triphenylphosphine dichloride (Ph₃PCl₂) (prepared from Ph₃P and Cl₂),trimethylsilylchloride and bismuth(III) trichloride (Me₃SiCl/BiCl₃),mixtures of Ph₃P and carbon tetrachloride (CCl₄), or methanesulfonylchloride (MeSO₂Cl) in pyridine at elevated temperatures.

Referring to Scheme 4, it will be appreciated by one skilled in the artthat the presence of particular functional or protecting groups incompounds of Formula (K-N) determines the choice a particular reagent,method, or reaction condition for the chloro-de-hydroxylation reaction.

Referring to Scheme 4, in certain embodiments R¹ and/or R⁵, R²⁰, thelinker L, E, the protecting groups PG and Q are defined as describedherein; one of R², R³, and R⁴ in compounds of Formula (K) and of Formula(L) is a -E-N,N-bis(2-hydroxyethyl)amino group (-E-N(CH₂—CH₂—OH)₂); eachof the other remaining R², R³, and R⁴ is hydrogen; and each R⁷ and eachR⁸ is hydrogen.

Referring to Scheme 4, in some embodiments N,N-bis(2-hydroxyethyl)compounds of Formula (K) or of Formula (L) may be reacted with an excessof about 2-15 equivalents of thionyl chloride (SOCl₂) either in neatform or as a solution in an anhydrous organic solvent, e.g.,dichloromethane (DCM), chloroform (CHCl₃), 1,2-dichloroethane (DCE),benzene, or mixtures of any of the foregoing at temperatures from about0° C. (ice bath) −40° C. or heated at reflux for about 0.5-3 hours toprovide compounds of Formula (M) or of Formula (N) (Palmer, et al., J.Med. Chem. 1990, 33(1), 112-121; Jordan, et al., Bioorg. Med. Chem.,2002, 10(8), 2625-2633; Abela Medici, et al., J. Chem. Soc., PerkinTrans. 1, 1997, (20), 2258-2263; Taylor, et al., Chem. Biol. Drug Des.,2007, 70(3), 216-226; Dheyongera, Bioorg. Med. Chem. 2005, 13(3),689-698; Zheng, Bioorg. Med. Chem. 2010, 18(2), 880-886; Gourdi, J. Med.Chem., 1990, 33(4), 1177-1186; and Lin, et al., Bioorg. Med. Chem.Lett., 2011, 21(3), 940-943). The reaction may optionally be carried outin the presence of a catalytic amount of zinc chloride (ZnCl₂) (10 mol-%to 40 mol-%) or in the presence of a catalytic amount ofN,N-dimethylformamide (DMF) to facilitate the reaction (Squires, et al.,J. Org. Chem., 1975, 40(1), 134-136; and Abela Medici, et al, J. Chem.Soc., Perkin Trans. 1, 1997, (20), 2258-2263).

Referring to Scheme 4, in some embodiments N,N-bis(2-hydroxyethyl)compounds of Formula (K) or of Formula (L) may also be reacted with anexcess of about 2-10 equivalents of phosphorus(V)oxychloride (phosphorylchloride, POCl₃) either in neat form or as a solution in an anhydrousorganic solvent, e.g., benzene, acetonitrile, pyridine, or mixtures ofany of the foregoing at a temperature from about 0° C. (ice bath) toabout room temperature. The reaction mixture may also be heated fromabout 80° C. to about reflux temperature for about 0.5-6 hours toprovide compounds of Formula (M) or of Formula (N) (Palmer, et al., J.Med. Chem. 1990, 33(1), 112-121; Feau, et al., Org. Biomolecular Chem.,2009, 7(24), 5259-5270; Valu, et al., J. Med. Chem., 1990, 33(11),3014-3019; Baraldini, et al., J. Med., Chem., 2000, 53(14), 2675-2684;Gourdi, et al., J., Med., Chem., 1990, 33(4), 1177-1186; Haines, et al.,J. Med. Chem., 1987, 30, 542-547; and Matharu, et al., Bioorg. Med.Chem. Lett., 2010, 20, 3688-3691).

Referring to Scheme 4, in some embodiments N,N-bis(2-hydroxyethyl)compounds of Formula (K) or of Formula (L) may also be reacted with anexcess of carbon tetrachloride (CCl₄), optionally in an inert solvent,e.g., dichloromethane (DCM), in the presence of an excess oftriphenylphosphine (Ph₃P) for about 8-24 hours at about room temperatureor at reflux temperature for about 2-6 hours to provide compounds ofFormula (M) or of Formula (N) (Buss, et al., J. Fluorine Chem., 1986,34(1), 83-114; and Kupczyk-Subotkowska, et al., J. Drug Targeting, 1997,4(6), 359-370).

Referring to Scheme 4, in some embodiments N,N-bis(2-hydroxyethyl)compounds of Formula (K) or of Formula (L) may also be reacted withmethanesulfonyl chloride (MeSO₂Cl, MsCl) in anhydrous pyridine at aboutroom temperature or at about 70-100° C. for about 1-3 hours to providecompounds of Formula (M) or of Formula (N) (Jordan, et al., Bioorg. Med.Chem., 2002, 10(8), 2625-2633; Abela Medici, et al, J. Chem. Soc.,Perkin Trans. 1, 1997, (20), 2258-2263; Springer, et al., J. Med. Chem.,1990, 33(2), 677-681; and Larden and H. T. A. Cheung, Tetrahedron Lett.,1996, 37(42), 7581-7582).

Referring to Scheme 5, although halides are common leaving groups innucleophilic substitution reactions for synthetic purposes, it is oftenmore convenient to use the corresponding alcohols such as the ones foundin N,N-bis(2-hydroxyethyl)amino groups of compounds of Formula (O).Since OH is usually considered a poor leaving group, unless protonated,conversion of a hydroxy group such as in N,N-bis(2-hydroxyethyl)aminogroups of compounds of Formula (O) into reactive ester groups, mostcommonly sulfonic ester groups, converts the hydroxyl group into afunctional group with a higher susceptibility to be displaced by anincoming nucleophile including halogenide ions. The N,N-bis(2-aryl- or(polyfluoro)alkylsulfonyloxy)amino groups of aryl- or(polyfluoro)alkylsulfonates of Formula (P) and similar sulfonic estersare most frequently prepared from N,N-bis(2-hydroxy)amino groups ofdiols of Formula (O) through reaction with an appropriate aryl- or(polyfluoro)alkylsulfonyl chloride or anhydride in the presence of asuitable base, e.g., pyridine (nucleophilic catalyst). Besides aromatic(R⁴⁰ is (substituted) aryl) sulfonic ester groups, aliphatic (R⁴⁰ isalkyl) sulfonic ester groups, and, in particular, (poly)fluorinated (R⁴⁰is poly-F-alkyl) sulfonic ester groups as still more powerful leavinggroups are frequently used for activation.

Referring to Scheme 5, in certain embodiments the R⁴⁰-group in compoundsof Formula (P) or Formula (R) is for example phenyl and the leavinggroup is phenylsulfonyloxy (PhSO₂O), 4-methylphenyl (para-methylphenyl)and the leaving group is tosylate (4-methylphenylsulfonyloxy, TsO),4-bromophenyl (para-bromophenyl) and the leaving group is brosylate(4-bromophenylsulfonyloxy, BsO), or 4-nitrophenyl (para-nitrophenyl) andthe leaving group is nosylate (4-nitrophenylsulfonyloxy, NsO), methyland the leaving group is mesylate (methanesulfonyloxy, MsO),trifluomethyl and the leaving group is triflate(trifluoromethanesulfonyloxy, TfO), nonafluoro-n-butyl and the leavinggroup is nonaflate (nonafluorobutanesulfonyloxy), or2,2,2-trifluoroethyl and the leaving group is tresylate(2,2,2-trifluoroethanesulfonyloxy). In some embodiments, the R⁴⁰-groupof compounds of Formula (P) and Formula (R) is methyl and the leavinggroup is mesylate (methansulfonyloxy, MsO). In some embodiments, theR⁴⁰-group of compounds of Formula (P) and of Formula (R) istrifluoromethyl and the leaving group is triflate(trifluoromethansulfonyloxy, TfO).

Referring to Scheme 5, N-mustard-type halides of Formula (Q), Formula(R), and Formula (S) containing either (a) aN,N-bis(2-halogenoethyl)amino group (compounds of Formula (Q)), (b) aN-(2-halogenoethyl)amino-, N-(2-halogeno′ethyl)amino-group (compounds ofFormula (S) or mixed halogeno N-mustards), or (c) aN-(2-halogenoethyl)amino, N-(2-aryl- or(polyfluoro)alkylsulfonyloxyethyl)amino groups (compounds of Formula (R)or hybrid halogeno sulfonate N-mustards), may be prepared from thecorresponding esters of sulfonic acid esters of Formula (P) throughreaction with an excess or a near stoichiometric amount of an alkalimetal halide (MX, MX′) in suitable protic or non-protic organic solventat elevated temperature (halo-de-sulfonyloxy substitution)

Referring to Scheme 5, in certain embodiments M in MX or MX′ is analkali metal cation, e.g., lithium (Li⁺) and sodium (Na⁺), X and X′ inMX or MX′ are halide anions, e.g., chloride (Cl⁻), bromide (Br⁻), andiodide (I⁻). MX or MX′ are alkali metal halides, e.g., lithium chloride(LiCl), lithium bromide (LiBr), sodium chloride (NaCl), sodium bromide(NaBr), or sodium iodide (NaI). In compounds of Formula (Q), Formula(R), and Formula (S), X is a halogeno, e.g., chloro (—Cl), bromo (—Br),and iodo (—I) (Palmer, et al., J. Med. Chem. 1990, 33(1), 112-121;Palmer, et al., J. Med. Chem., 1994, 37, 2175-2184; Palmer, et al., J.Med. Chem., 1996, 39(13), 2518-2528; Davies, et al., J. Med. Chem. 2005,48(16), 5321-5328; Niculesscu-Duvaz, et al., J. Med. Chem., 2004,47(10), 2651-2658; Weisz, et al., Bioorg. Med. Chem. Lett., 1995, 5(24),2985-2988; Thorn, J. Org. Chem, 1975, 40(11), 1556-1558; Lin, et al.,Bioorg. Med. Chem. Lett., 2011, 21(3), 940-943; Gourdi, et al., J. Med.Chem. 1990, 33(4), 1177-1186; Yang, et al., Tetrahedron, 2007, 63(25),5470-5476; Ferlin, et al., Bioorg. Med. Chem., 2004, 12(4), 771-777; andCoggiola, et al., Bioorg. Med. Chem. Lett., 2005, 15(15), 3551-3554).

Referring to Scheme 5, N-(2-halogenoethyl)amino, N-(2-aryl- oralkylsulfonyloxyethyl)amino groups of Formula (R) (hybrid halogenosulfonate N-mustards) may also be prepared from primary alkyl halides ofFormula (Q) containing N,N-bis(2-halogenoethyl)amino groups through a) ahalo-de-halogenation (halide exchange reaction) or b) a metatheticalsulfonyloxy de-halogeno substitution reaction with solubilized silversulfonates AgOSO₂R⁴⁰ wherein R⁴⁰ is defined as described herein undermild conditions in aprotic organic solvents (Emmons and Ferris, J Am.Chem. Soc., 1953, 75(9), 2257).

Referring to Scheme 5, it will be obvious to those skilled in the artthat comparable reagents and reaction conditions may be used tointroduce a) the N,N-bis(2-aryl- or (polyfluoro)alkyl-sulfonyloxyethyl)functionality, b) the N,N-bis(2-halogenoethyl) functionality, c) theN-(2-halogenoethyl)N-bis(2-halogeno′ethyl) functionality, d) theN-(2-aryl- or (polyfluoro)alkyl-sulfonyloxyethyl)N-(2-halogenoethyl), orderivatives of any of the foregoing, using the corresponding internallyprotected β-substituted γ-amino acid precursors (γ-lactams) described inSchemes 2-4.

Referring to Scheme 5, for example in certain embodiments R¹ and/or R⁵,R²⁰, R⁴⁰, X, X′, E, the linker L, the protecting groups PG and Q aredefined as herein; one of R², R³, and R⁴ in compounds of Formula (O) is-E-N(CH₂—CH₂—OH)₂ each of the other remaining R², R³, and R⁴ ishydrogen; and each of R⁷ and R⁸ is hydrogen.

Referring to Scheme 5, in certain embodiments, theN,N-bis(2-hydroxyethyl)amino group of compounds of Formula (O) may beconverted to N,N-bis(2-(polyfluoro)alkyl- or arylsulfonyloxyethyl)aminogroups of compounds of Formula (P) (S-alkoxy-de-chlorination) byreacting diols of Formula (O) with an excess of a suitable(perfluoro)alkyl- or aryl-sulfonyl anhydride (R⁴⁰SO₂)₂O) (about 2.5-5equivalents), e.g., methanesulfonyl anhydride (R⁴⁰=methyl (Me),(MeSO₂)₂O)), in an inert solvent such anhydrous dichloromethane (DCM) ortetrahydrofuran (THF) or a mixture of any of the foregoing in thepresence of an excess (about 2-10 equivalents) of a suitable base, e.g.,anhydrous triethylamine (Et₃N, TEA) or anhydrous pyridine, at atemperature from about 0° C. to about room temperature for about 0.5-24hours to afford bis-sulfonic acid esters of Formula (P). The reactionmay optionally be carried out in the presence of a catalytic amount(about 20 mol-%) of 4-N,N-(dimethylamino)pyridine (DMAP).

Referring to Scheme 5, in certain embodiments, using comparable reactionconditions with respect to solvents, bases, stoichiometry of reagents,temperature, catalysts, and duration as described for the reaction ofdiols of Formula (O) with (polyfluoro)alkyl- or aryl-sulfonylanhydrides, diols of Formula (O) may also be reacted with a suitablealkyl- or aryl-sulfonyl halides, e.g., methanesulfonyl chloride (mesylchloride, MsCl) (R⁴⁰=Me), MeSO₂Cl), to provide the desired bis-sulfonicacid esters of Formula (P).

Referring to Scheme 5, in certain embodimentsN,N-bis(2-(polyfluoro)alkyl- or aryl-sulfonyloxyethyl)amino groups as incompounds of Formula (P) may be converted (halo-de-sulfonyloxysubstitution) to N,N-bis(halogenoethyl)amino groups of compounds ofFormula (Q) by reacting bis-sulfonyl esters of Formula (P) with anexcess of a suitable alkali metal halide salt MX, e.g., lithium chloride(LiCl), lithium bromide (LiBr), sodium chloride (NaCl), sodium bromide(NaBr), or sodium iodide (NaI) (4-16 equivalents) in a suitable organicsolvent, e.g., N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMAc), acetone, 2-butanone (methyl ethyl ketone, MEK),3-methyl-2-butanone (isopropyl methyl ketone, MIPK), acetonitrile(MeCN), methanol (MeOH), tetrahydrofuran (THF), ethyl acetate (EtOAc),or a mixture of any of the foregoing, at room temperature or heated toabout 50-150° C. for about 0.5-6 hours to provide compounds of Formula(Q).

Referring to Scheme 5, in certain embodiments using comparable reactionconditions with respect to solvents, temperature, and duration asdescribed for the preparation of compounds of Formula (Q), the reactionof bis-sulfonyl esters of Formula (P) may also be carried out in thepresence of about one molar equivalent of a suitable alkali metal halidesalt MX, as defined herein, to provide compounds of Formula (R) bearingN-(2-halogenoethyl)-, N-(2-methyl sulfonyloxyethyl) amino groups (mixedhalogeno/sulfonylato N-mustards).

Referring to Scheme 5, in some embodiments compounds of Formula (Q) maybe converted to mixed halogeno/sulfonylato N-mustards of Formula (R) byreacting N-mustard derivatives of Formula (Q) where X is bromo (—Br)with about 1.0 equivalent or slightly less of a suitable soluble silversulfonate salt, e.g., silver mesylate (AgOSO₂Me, AgOMs) in a polarsolvent such as acetonitrile (MeCN) at about reflux temperature toprovide the mixed halogeno/mesylate N-mustard of Formula (S)(methathetical reaction).

Referring to Scheme 5, in certain embodiments, using comparable reactionconditions with respect to solvents, temperature, and duration asdescribed for the preparation of compounds of Formula (Q) and of Formula(R), the reaction of bis-halogeno N-mustards of Formula (Q) or of mixedhalogeno/mesylate N-mustards of Formula (R) may also be carried out inthe presence of about one molar equivalent of a suitable alkali metalhalide salt MX′, as defined herein, to provide compounds of Formula (S)bearing N-(2-halogenoethyl)-, N-(2-halogeno′ethyl) amino groups (mixedhalogeno N-mustards).

Reductive N-alkylation is a form of amination/alkylation that involvesthe reaction of an amino group with a carbonyl group to an amine in thepresence of a suitable reducing agent via an intermediate imine orprotonated imine. The carbonyl group component is most commonly analdehyde or ketone functionality, the amino group is most commonlyammonia, a primary or secondary aliphatic amino group, or a primary orsecondary aromatic amino group (aniline). For indirect reductiveaminations, the intermediate imine may be isolated and reduced with asuitable reducing agent. For direct reductive aminations, the reactionmay be carried out simultaneously, with the imine formation andreduction occurring concurrently, typically using reducing agents thatare more reactive toward protonated imines than ketones, and that arestable under moderately acidic conditions, e.g., sodium cyanoborohydride(Na(CN)BH₃) or sodium triacetoxyborohydride (NaB(OAc)₃H.

Referring to Scheme 6, the primary amino group of compounds of Formula(T) or of Formula (U), either in a suitable salt form, e.g., ahydrochloride (HCl) salt (Ar-E-NH₂.HCl) or as a free base (Ar-E-NH₂) maybe subjected to a reductive N-alkylation reaction using a suitablehalocarbonyl compounds (X=F, Cl or, Br) or derivatives thereof, e.g., adimethyl acetal, and reducing agents as they are well known in the art(Palani, et al., J. Med. Chem., 2005, 48(15), 4746-4749; Van Oeveren,Bioorg. Med. Chem. Lett., 2007, 17(6), 1527-1531; Delfourne, et al.,Bioorg. Med. Chem., 2004, 12(15), 3987-3994; Delfourne, et al., J. Med.Chem., 2002, 47(17), 3765-3771; and Jordan, et al., Bioorg. Med. Chem.,2002, 10(8), 2625-2633).

Suitable halocarbonyl compounds include, for example, 2-chloroaceticacid (ClCH₂CO₂H, X is Cl)), 2-chloroacetaldehyde (ClCH₂CHO, X is Cl)),or 2-bromoacetaldehyde dimethylacetal (MeO)₂CHCH₂Br, X is Br),optionally provided as solutions in suitable solvents, e.g., a 50-wt-%solution of 2-chloroacetaldehyde (ClCH₂CHO, X is Cl)) in water.

Referring to Scheme 6, suitable reducing agents for reductiveN-alkylations of primary amino groups such as in compounds of Formula(T) and of Formula (U) using 2-chloroacetic acid include boranes,preferably borane-tetrahydrofuran complex (H₃B.THF), and ceratinalkalimetal borohydrides, e.g., lithium borohydride (LiBH₄) or sodiumborohydride (NaBH₄).

Referring to Scheme 6, the reaction is generally carried out in thepresence of organic solvents such as protic solvents, e.g., methanol(MeOH), acetic acid, (HOAc), trifluoroacetic acid (TFA), 85 wt-%phosphoric acid (H₃PO₄), glacial acetic acid (HOAC), 98 wt-% formicacid, or water, or inert organic solvents, e.g., acetonitrile (MeCN),dichloromethane (DCM), tetrahydrofuran (THF), benzene, or equivalentmixtures of any of the foregoing at a temperature from about 0° C. toabout reflux temperature and for about 05-18 hours. In embodiments where2-chloroacetaldehyde is used, suitable reducing agents may include, forexample, sodium cyanoborohydride (Na(CN)BH₃), sodiumtriacetoxyborohydride (NaB(OAc)₃H, and sodium borohydride (NaBH₄).

Reduction via hydrogenation is can also be employed. Preferredhydrogenation conditions include catalytic hydrogenation, for example,using palladium on carbon (Pd/C) as the catalyst. As the hydrogensource, gaseous hydrogen (H₂-gas) at pressures ranging from aboutatmospheric pressure to about 150 psi, or suitable ammonium salts, e.g.,ammonium hydrogencarbonate (H₄NHCO₃), may be employed. The hydrogenationmay be carried out at ambient temperature.

Referring to Scheme 6, in certain embodiments, R¹ and/or R⁵, R²⁰, E, thelinker L, the halogeno group X, and the protecting group PG and Q aredefined as herein; one of R², R³, and R⁴ in compounds of Formula (T) andFormula (U) is -E-NH₂, wherein E is a bond (“-”), an oxygen atom (—O—),a methylene group (—CH₂—), or methylenoxy group (—CH₂—O—), and whereinMH is an amino group (—NH₂) so that -E-NH₂ is equivalent to a) a primaryaromatic amino group (—NH₂, aniline), b) a primary O-aryl hydroxylaminogroup (—O—NH₂), c) a primary aminomethyl group (—CH₂—NH₂), or a primaryO-benzyl hydroxylamino group (—CH₂—O—NH₂); each of the other remainingR², R³, and R⁴ is hydrogen; each of R⁷ and R⁸ is hydrogen.

Referring to Scheme 6, in certain embodiments, the primary amino groupof compounds of Formula (T) or of Formula (U) may be converted toN,N-bis(2-halogenoethyl)amino groups as in compounds of Formula (V) orof Formula (W) by reacting compounds of Formula (T) or of Formula (U)with an excess of about 4-10 equivalents of a 2-halogenocarbonylcompound, e.g., a 50 wt-% solution of 2-chloroacetaldehyde in water, andan excess of about 3-8 equivalents of a suitable reducing agent, e.g.,sodium cyanoborohydride (NaB(CN)H₃). In certain embodiments, thereaction may be carried out in mixtures of methanol (MeOH) withtrifluoroacetic acid (TFA), glacial acetic acid (HOAc), 98 wt-% formicacid (FA), or 85 wt-% phosphoric acid (H₃PO₄). For example, in certainembodiments, 1:1 (v/v), 2:1 (v/v), or 1:2 (v/v) mixtures MeOH/acid andreaction temperatures from about 0-40° C. and reaction times of about0.5-18 hours are employed to provide protected N-mustards of Formula (V)or of Formula (W).

Estramustine (Emcyt®, Estracit®) is an antimicrotubule chemotherapyagent indicated in the United States for the palliative treatment ofmetastatic and/or progressive prostate cancer. It is derivative ofestrogen (specifically, estradiol) with a N-mustard-carbamate estermoiety.

Referring to Scheme 7, methods to functionalize alcohols or phenols withcarbamoyl derivatives of secondary amines yielding carbamates as in, forexample, compounds of Formula (Z) wherein M is oxygen (—O—) and G isoxygen (═O) include carbamoyl chlorides or p-nitrophenyl carbamates, andare well known in the art. Likewise, it is well known in the art thatcarbamates as in, for example, compounds of Formula (Z) wherein M isoxygen (—O—) and G is oxygen (═O) are also accessible through activationof alcohols or phenols with suitable formic ester derivatives includingphosgene (COCl₂), triphosgene (bis(trichloromethyl) carbonate (BTC)), or1,1′-carbonyldiimidazole (CDI) followed by reaction with anappropriately functionalized amine such as HN(CH₂—CH₂—R⁹)₂ wherein R⁹ ischloro (—Cl), bromo (—Br), iodo (—I), or (polyfluoro)alkyl- or arylsulfonyloxy (—OSO₂R⁴⁰) or combinations thereof and R⁴⁰ is defined asdescribed herein.

Likewise and referring to Scheme 7, many methods are known in theliterature and to those skilled in the art to prepare compounds ofFormula (Z) related to carbamates including a) S-thiocarbamates whereinM is sulfur (—S—) and G is oxygen (═O), b) O-thiocarbamates wherein M isoxygen (—O—) and G is sulfur (═S), c) dithiocarbamates wherein M issulfur (—S—) and G is sulfur (═S), d) ureas wherein M is nitrogen(—NR¹⁰—), and where R¹⁰ is defined as described herein, and G is oxygen(═O), or thioureas wherein M is nitrogen (—NR¹⁰—) and G is sulfur (═S).

Referring to Scheme 7, in certain embodiments a compound of Formula (X)is, for example, a) a phenol wherein E is a bond (“-”) and MH is ahydroxyl group (—OH), b) an aniline wherein E is a bond (“-”) and MH isan amino group (—NR¹⁰H), c) a thiophenol wherein E is a bond (“-”) andMH is a sulfhydryl group (—SH), d) an O-aryl hydroxylamine wherein E isoxygen (—O—) and MH is an amino group (—NR¹⁰H), e) a benzylic alcoholwherein E is methylene (—CH₂—) and MH is a hydroxyl group (—OH), f) abenzylic amine wherein E is methylene (—CH₂—) and MH is an amino group(—NR¹⁰H), g) a benzylic thiol wherein E is methylene (—CH₂—) and MH issulfhydryl (—SH), h) an O-benzylic hydroxylamine wherein E ismethyleneoxy (—CH₂—O—) and MH is an amino group (—NR¹⁰H).

Referring to Scheme 7, in certain embodiments, R¹ and/or R⁵, R¹⁰, R²⁰,E, M, Z, the linker L, and the protecting group PG and Q are as definedherein; one of R², R³, and R⁴ in compounds of Formula (X) is -E-MH asdescribed herein; each of the other remaining R², R³, and R⁴ ishydrogen; each R⁷ and each R⁸ is hydrogen; LG is a suitable leavinggroup such as to chloro (—Cl), 4-nitrophenyloxy (NO₂C₆H₄O—), orimidazole; and R⁹ is chloro (—Cl), bromo (—Br), iodo (—I), or(polyfluoro)alkyl- or aryl sulfonyloxy (—OSO₂R⁴⁰) or combinationsthereof, and R⁴⁰ is defined as described herein.

Referring to Scheme 7, in certain embodiments the alcohol, the thiolgroup, or the amino group of compounds of Formula (X) may be convertedto the N,N-bis(2-halogeno- or 2-sulfonyloxyethyl)carbamoyl orN,N-bis(2-halogeno- or 2-sulfonyloxyethyl)thiocarbamoyl group ofcompounds of Formula (Z) by reacting a compound of Formula (X) with, forexample, commercial N,N-bis(2-chloroethyl)carbamoyl chloride (U.S. Pat.No. 3,299,104), wherein LG is chloro (—Cl), R⁹ is chloro (—Cl), and G isoxygen (═O) or known (4-nitrophenyl) N,N-bis(2-chloroethyl)carbamatewhere LG is 4-nitrophenol (4-NO₂-Ph-O—), R⁹ is chloro (—Cl), and G isoxygen (═O) in suitable solvents such as pyridine, or triethylamine in1,4-dioxane/benzene mixtures and the like at temperatures of about 0-60°C. to provide carbamate, thiocarbamate, or urea derivatives of Formula(Z).

Referring to Scheme 7, in certain embodiments the MH-group of compoundsof Formula (X) may be activated to their corresponding chloroformates,thiochloroformates, or carbonyl imidazoles of Formula (Y) with, forexample, phosgene, thiosphosgene, triphosgene, carbonyldiimidazole(CDI), thiocarbonyldiimidazole (TCDI), or the like, in the presence of asuitable base such as inorganic metal-carbonate, e.g., potassiumcarbonate (K₂CO₃) and bicarbonates, e.g., sodium hydrogencarbonate(NaHCO₃), in suitable inert solvents known in the art. Thechloroformates or thiochloroformates of Formula (Y) are subsequentlyconverted to the corresponding carbamates of Formula (Z) throughreaction with an appropriately functionalized amine such asHN(CH₂—CH₂—R⁹)₂ wherein R⁹ is chloro (—Cl), bromo (—Br), iodo (—I), or(polyfluoro)alkyl- or aryl sulfonyloxy (—OSO₂R⁴⁰) or combinationsthereof, and R⁴⁰ is defined as described herein, e.g., commercialbis(2-chloroethyl)amine hydrochloride wherein R⁹ is chloro (—Cl) or2-bromo-N-(2-bromoethyl)ethanamine wherein R⁹ is bromo (—Br), and in thepresence of a base such as inorganic metal-carbonate, e.g., potassiumcarbonate (K₂CO₃) and bicarbonate, e.g., sodium hydrogencarbonate(NaHCO₃), ethyl acetate (EtOAc), water, or mixtures of any of theforegoing to yield carbamates of Formula (Z).

In general, the biological activity of nitrogen mustards is based uponthe presence of an alkylating N,N-bis(2-chloroethyl) functionality. Thechemotherapeutic and cytotoxic effects are directly associated with thealkylation of DNA due to the strong electrophilic character of theN,N-bis(2-chloroethyl) functionality. Formation of covalent linkagesincluding interstrand crosslinks (ICLs) is highly cytotoxic and involvesthe disruption of fundamental cellular processes including DNAreplication leading to cellular death.

Because of this property, the nitrogen mustards have been used for anumber of years in laboratory investigations and in the clinical treatfor malignat growth. Unfortunately, the effective dose of nitrogenmustards is in many cases close to the toxic dose and it is thereforedesirable to find a nitrogen mustard or a class of nitrogen mustard typecompounds possessing the high carcinolytic activity of the parentcompound but having modulated toxicity.

The amide linkage masks the alkylating and toxic properties of thenitrogen mustard moiety so that the total host is not subjected toundesirable toxic effects sometime encountered with nitrogen mustardtherapy: the amino acid moiety of the molecule facilitates the selectivedelivery of the “masked” nitrogen mustard via the amino acid transportmechanism into the tumor cells, where the higher amidase activity of thetumor cell liberates the reactivated nitrogen mustard within itself.Thus, in effect it will be possible to obtain maximum effect of thenitrogen mustard on the tumor and minimum toxic effect on the host (U.S.Pat. No. 3,235,594).

Referring to Scheme 8, the amide nitrogen mustards of the presentdisclosure are prepared by condensing carboxylic acids of Formula (AA)wherein E is a carbonyl group (—C(═O)—) or a methylenecarbonyl group(—CH₂—C(═O)—) with an appropriately functionalized amine such asHN(CH₂—CH₂—R⁹)₂ wherein X is chloro (—Cl), bromo (—Br), iodo (—I), or(polyfluoro)alkyl- or aryl sulfonyloxy (—OSO₂R⁴⁰) or combinationsthereof, and R⁴⁰ is defined as described herein, to provide amides ofnitrogen mustards of Formula (AB).

Referring to Scheme 8, a myriad of coupling methods is known in the artto facilitate the formation of amide bonds as in compounds of Formula(AB) from carboxylic acids of Formula (AA) (Montalbetti and Falque,Tetrahedron, 2005, 61, 10827-10852; and Valeur and Bradley, Chem. Soc.Rev., 2009, 38, 606-631).

Refering to Scheme 8, in certain embodiments, R¹ and/or R⁵, R²⁰, E, thelinker L, and the protecting group PG and Q are defined as describedherein; one of R², R³, and R⁴ in compounds of Formula (AA) is -E-OH asdescribed herein; each of the other remaining R², R³, and R⁴ ishydrogen; each R⁷ and each R⁸ is hydrogen; R⁹ is a suitablefunctionalization providing the alkylation properties of the nitrogenmustard.

Referring to Scheme 8, in certain embodiments the (thio)carboxyl groupof compounds of Formula (AA) may be activated as acyl halides, acylazides, symmetrical or unsymmetrical carboxylic, carbonic, or boronicanhydrides, acyl imidazoles, activated esters, phosphonium salts,uronium salts, or ammonium salts followed by ammonolysis of theactivated intermediate either after prior isolation or in situ with anappropriately functionalized amine such as HN(CH₂—CH₂—R⁹)₂ to providenitrogen mustard amides of Formula (AB).

Referring to Scheme 9, protected N-mustard functionalized β-substitutedγ-amino acid precursors or protected N-mustard functionalizedβ-substituted γ-amino acid analog precursors or carboxylic acid(bio)isosteres of Formula (AC) or of Formula (AD) are converted to thecorresponding unprotected N-mustard functionalized β-substituted γ-aminoacid derivatives or unprotected N-mustard functionalized β-substitutedγ-amino acid analogs or carboxylic acid (bio)isosteres of Formula (AE)by removing the external protecting groups and/or by opening the lactamring (internal protection).

Referring to Scheme 9, in certain embodiments the connector group “A” ofthe moiety -A-N(CH₂—CH₂—R⁹)₂ is a bond (“-”), oxygen (—O—), sulfur(—S—), methylene (—CH₂—), methyleneoxy (—CH₂—O—), oxycarbonyl(—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl (—NR¹⁰—C(═O)—),oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl (—S—C(═S)—),aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl (—CH₂—O—C(═O)—),methylenethiocarbonyl (—CH₂—S—C(═O)—), methyleneaminocarbonyl(—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl (—CH₂—O—C(═S)—),methylenethiothiocarbonyl (—CH₂—S—C(═S)—), methyleneaminothiocarbonyl(—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—), methylencarbonyl (—CH₂—C(═O)—),thiocarbonyl (—C(═S)—), methylenthiocarbonyl (—CH₂—C(═S)—).

Referring to Scheme 9, in certain embodiments liberation of unprotectedN-mustard functionalized β-substituted γ-amino acid derivatives orunprotected N-mustard functionalized β-substituted γ-amino acid analogsor carboxylic acid (bio)isosteres of Formula (AE) from theircorresponding precursors of Formula (AC) or of Formula (AD) may beconducted under aqueous acidic conditions (hydrolysis) (Taylor, et al.,Chem. Biol. Drug Des., 2007, 70(3), 216-226; Buss, et al., J. FluorineChem., 1986, 34(1), 83-114; Abela, et al, J. Chem. Soc., Perkin Trans.1, 1997, (20), 2258-2263; Weisz, et al., Bioorg. Med. Chem. Lett., 1995,5(24), 2985-2988; Zheng, Bioorg., Med., Chem., 2010, 18(2), 880-886;Haines, et al., J. Med. Chem., 1987, 30, 542-547; and Matharu, et al.,Bioorg., Med., Chem., Lett., 2010, 20, 3688-3691).

Referring to Scheme 9, in certain embodiments liberation of unprotectedN-mustard functionalized β-substituted γ-amino acid derivatives orunprotected N-mustard functionalized β-substituted γ-amino acid analogsor carboxylic acid (bio)isosteres of Formula (AE) from theircorresponding precursors of Formula (AD) may also be conducted underanhydrous acidic conditions (Springer, et al., J. Med. Chem., 1990,33(2), 677-681; Davies, et al., J. Med. Chem. 2005, 48(16), 5321-5328;Niculesscu-Duvaz, et al., J. Med. Chem., 2004, 47(10), 2651-2658; Vernyand Nicolas, J. Label. Cmpds, Radiopharm., 1988, 25(9), 949-955; Thorn,et al., J. Org. Chem, 1975, 40(11), 1556-1558; Baraldini, et al., J.Med. Chem., 2000, 53(14), 2675-2684; Gourdi, et al., J. Med. Chem.,1990, 33(4), 1177-1186; and Kupczyk-Subotkowska, et al., J. DrugTargeting, 1997, 4(6), 359-370).

Referring to Scheme 9, it will be obvious to those skilled in the artthat protected N-mustard functionalized β-substituted γ-amino acidprecursors of Formula (AA) or protected N-mustard β-substituted γ-aminoacid analog or carboxylic acid (bio)isosteres precursors of Formula (AD)bearing different combinations of suitable protecting groups may also beprepared. Different combinations of protecting groups may requirespecific reactants and reaction conditions for effective removal ofspecific set of different protection groups to provide unprotectedN-mustard β-substituted γ-amino acid derivatives or unprotectedN-mustard funtionalized β-substituted γ-amino acid derivatives, analogs,or carboxylic acid (bio)isosteres of Formula (AE).

Referring to Scheme 9, in certain embodiments of compounds of Formula(AC), Formula (AD), and of Formula (AE) R¹ and/or R⁵, R⁹, the connectorgroup A, the protecting groups PG and Q, and the linker L are defined asdescribed herein; R⁶ is an unprotected carboxylic acid, a carboxylicacid analog or a carboxylic acid (bio)isostere as defined herein; R²⁰ isa protected carboxylic acid, a carboxylic acid analog or a carboxylicacid (bio)isostere as defined herein; one of R², R³, and R⁴ is aN,N-bis-(2-functionalized)ethylamino group (nitrogen mustard group)linked to a connector A (-A-N(CH₂—CH₂—R⁹)₂); each of the remaining R²,R³, and R⁴ is hydrogen; each of R⁷ and R⁸ is hydrogen.

Referring to Scheme 9, hydrolytic acidic ring-opening and simultaneousinternal N-deprotection of compounds of Formula (AC) or hydrolyticacidic global deprotection of compounds of Formula (AD) to provideN-mustard functionalized β-substituted γ-amino acid derivatives orN-mustard functionalized β-substituted γ-amino acid analogs orcarboxylic acid (bio)isosteres of Formula (AE) may be accomplished bytreating protected precursors of Formula (AC) or of Formula (AD) atelevated temperatures from about 40-150° C. with aqueous mineral acids,e.g., 2 M to ˜12 M hydrochloric acid (HCl) for about 6-24 hours. Incertain embodiments, mixtures of the mineral acid with organic solventsmay be used. A useful aqueous mineral acid reaction mixture tofacilitate lactam ring opening or global deprotection is, e.g., a 1:1(v/v) mixture of concentrated hydrochloric acid (˜12M or ˜37 wt-% HCl)with 1,4-dioxane.

Referring to Scheme 9, other aqueous mineral acids with anon-nucleophilic anion known in the art can be used to facilitatesimultaneous hydrolytic acidic ring-opening and simultaneous internalN-deprotection of compounds of Formula (AC) or hydrolytic acidic globaldeprotection of acid-labile or hydrolysis sensitive protecting groups ofthe protected carboxylic moiety, of the protected carboxylic acid(bio)isostere, or of the amino functionality of compounds of Formula(AD) to provide N-mustard functionalized β-substituted γ-amino acidderivatives or N-mustard functionalized β-substituted γ-amino acidanalogs or carboxylic acid (bio)isosteres of Formula (AE).

Referring to Scheme 9, suitable mineral acids may include, for example,diluted or concentrated aqueous solutions of hydrobromic acid (HBr),hydroiodic acid (HI), sulfuric acid (H₂SO₄), perchloric acid (HClO₄),and phosphoric acid (H₃PO₄), mixtures of any of the foregoing ormixtures with suitable organic solvents, e.g., 1,4-dioxane, with any ofthe foregoing.

It is within the ability of one skilled in the art to select specificand suitable aqueous mineral acids and reaction conditions forhydrolytic acidic ring-opening and simultaneous internal N-deprotectionof compounds of Formula (AC) or hydrolytic acidic global deprotection ofcompounds of Formula (AD) to provide N-mustard functionalizedβ-substituted γ-amino acid derivatives or N-mustard functionalizedβ-substituted γ-amino acid analogs or carboxylic acid (bio)isosteres ofFormula (AE).

Referring to Scheme 9, simultaneous global deprotection of compounds ofFormula (AD) where R²⁰ is an acid labile moiety derived from acarboxylic acid, e.g., CO₂tBu, CO₂-pentamethylbenzyl,CO₂-(4-methoxy)benzyl, or CO₂-trityl, and Q is a protected amino groupderived from an acid-labile N-protecting group, e.g., N(H)Boc,N(H)trityl, N(H)(4-methoxy)phenyl-diphenylmethyl, orN(H)di-((4-methoxy)phenyl)-phenylmethyl, may also be accomplished byreaction with strong organic acids under anhydrous conditions toliberate free (unprotected)N-mustard functionalized β-substitutedγ-amino acid derivatives or N-mustard functionalized β-substitutedγ-amino acid analogs or carboxylic acid (bio)isosteres of Formula (AE).

In certain embodiments, strong (organic) acids useful for globaldeprotection under anhydrous conditions include trifluoroacetic acid(TFA), 98 wt-% formic acid (FA), methanesulfonic acid (MeSO₃H), 85 wt-%phosphoric acid (H₃PO₄), 2 M hydrogen chloride (HCl) in diethyl ether(Et₂O), 4 M hydrogen chloride (HCl) in 1,4-dioxane, or a saturatedsolution of HCl in ethyl acetate (EtOAc) (Li, et al., J. Org. Chem.,2006, 71, 9045-9050).

Depending of the overall sensitivity to strong (organic acids),compounds of Formula (AD) may be reacted with neat either neat strong(organic) acid or with solutions of the strong organic acid in suitableinert solvents such asdichloromethane (DCM), dichloroethane (DCE),1,4-dioxane, diethylether (Et₂O), tetrahydrofuran (THF), or toluenetypically in ratios ranging from neat (organic) acid to about 10 vol-%(organic) acid in said inert solvent, and reaction temperatures rangingfrom about 0-50° C. for about 1-24 hours to provide unprotectedN-mustard functionalized β-substituted γ-amino acid derivatives orunprotected N-mustard functionalized β-substituted γ-amino acid analogsor carboxylic acid (bio)isosteres of Formula (AE).

Optionally, 2-5 equivalents of a suitable scavenging agent such astriethysilane (Et₃SiH) (TES), triisopropylsilane (iPr₃SiH), thioanisole,or 1,2-dithioethane (HSCH₂CH₂HS) may be added to the reaction mixture tosuppress formation of unwanted side reactions and byproductsoriginating, for example, from alkylation of electron-rich aromaticscaffolds or sulfide groups under global deprotection conditionsdisclosed herein to provide unprotected N-mustard functionalizedβ-substituted γ-amino acid derivatives or unprotected N-mustardfunctionalized β-substituted γ-amino acid analogs or carboxylic acid(bio)isosteres of Formula (AE). Separation of unprotected N-mustardfunctionalized β-substituted γ-amino acid derivatives or unprotectedN-mustard functionalized β-substituted γ-amino acid analogs orcarboxylic acid (bio)isosteres of Formula (AE) from unreacted startingmaterials, unwanted byproducts, and impurities may be accomplishedusing, for example, solid-phase extraction (SPE) techniques, e.g., withQMA® cartridges (Waters, USA), LiChrolut® cartridges (EMD Chemicals,USA), or Whatman SAX cartridges (Whatman, USA), preparative normal orreverse phase TLC, reverse phase (RP) semi-preparative or preparativeHPLC, crystallization, precipitation, or any other suitable method knownin the art.

Purified unprotected N-mustard functionalized β-substituted γ-amino acidderivatives or unprotected N-mustard functionalized β-substitutedγ-amino acid analogs or carboxylic acid (bio)isosteres of Formula (AE)may be isolated using any of the methods known in the art. For example,such methods include removal of HPLC solvents (mobile phase) of thecombined fractions containing the N-mustard functionalized β-substitutedγ-amino acid derivatives or N-mustard functionalized β-substitutedγ-amino acid analogs or carboxylic acid (bioisosteres) of Formula (AE)under reduced pressure with a rotary evaporator, or removal of (aqueous)solvent mixtures by primary lyophilization.

Any suitable method known in the art may be used to produce acid aditionsalts or salts including pharmaceutically acceptable acid adition saltsor salts of compounds of Formula (AE).

The lyophilization may optionally be conducted in the presence of one ormore equivalents of a mineral acid, optionally with a pharmaceuticallyacceptable counterion, to form (pharmaceutically acceptable) acidaddition salts of compounds of Formula (AE). For example, one or moreequivalents of hydrochloric acid (HCl) may be added prior tolyophiliation to form mono-, di-, or polyhydrochloride salts ofcompounds of Formula (AE) or mixtures thereof.

The lyophilization may optionally be conducted in the presence of one ormore equivalents of a base, optionally with a pharmaceuticallyacceptable counterion, to form (pharmaceutically acceptable) salts ofcompounds of Formula (AE). For example, one or more equivalents ofsodium hydrogen carbonate (NaHCO₃) may be added prior to lyophiliationto form mono-, di-, or poly sodium salts of compounds of Formula (AE) ormixtures thereof.

A characteristic feature of solid tumors is the presence of cells atvery low oxygen concentrations (hypoxia; partial pressure of oxygen intumorous tissue of 0.05-5.0%) often surrounding areas of necrosis. Thereare clear links between hypoxia and the lack of response to radiotherapyand intrinsic resistance to cytotoxic therapy. It has also beendemonstrated that hypoxia in tumours tends to select for a moremalignant phenotype (Wilson and Hay, Nat. Rev. Canc., 2011, 11, 393-410;and Brown and Wilson, Nat. Rev. Canc., 2004, 4, 437-447).

Reductive metabolic processes are more prevalent in the hypoxicenvironment of solid tumors. Reductive enzyme systems have the abilityto reduce certain functional groups. For example, aromatic and aliphaticN-oxides (—N⁺(O⁻)R₂) are known to be reducible to the correspondingamines (—NR₂), and nitro groups (—NO₂) can be either reduced to thecorresponding amines (—NH₂) or to hydroxylamines (—NH(OH) depending onthe oxygen saturation of the tissue (Denny, et al., Br. J. Canc., 1996,74, Suppl. XXVII, S32-S38; and Nagasawa, et al., Biol. Pharm. Bull.,2006, 29(12), 2335-2342).

One promising approach for the design of cancer-cell-selective mustardsexploits selective enzymatic reduction of nitroaryl compounds in theoxygen-starved (hypoxic) cells found in solid tumors. N-Oxidederivatives of nitrogen mustards including N-oxides of melphalan(PX-478; U.S. Pat. No. 7,399,785; Koh, et al., Mol. Canc. Ther., 2008,7(1), 90-100; www.medkoo.com) and chlorambucil (Kirkatrick, et al.,Anti-Cancer Drugs, 1994, 5, 467-472; Tercel, et al., J. Med. Chem.,1995, 38, 1247-1252; and U.S. Pat. No. 5,602,273) have been investigatedas bioreductive prodrugs with reduced systemic toxicity in comparison tothe parent drugs. Those drugs take advantage of a) the hypoxic nature,and b) the reductive nature, of certain tumorous cells. The N-oxidefunctional group deactivates the extremely reactive alkylating agentthrough capture of the lone electron pair of the parent nitrogen mustardmoiety thus diminishing the alkylating properties and the off-targettoxicityies associated with that. Bioreductive activation within thehypoxic tumor environment or milieu by hypoxic cells and their reductiveenzyme systems is believed to restore the cytotoxicity of the freenitrogen mustards. The overall effect is an enhanced therapeutic indexof the N-oxides of nitrogen mustards relative to their parent nitrogenmustards.

Depending on the pH and the nature of the solvent, particularly aproticorganic solvents, N-oxides of nitrogen mustards are known tointramolecularly rearrange to the corresponding more stablehydroxylamines with markedly less intrinsic cytotoxic potential (Tercel,et al., J. Med. Chem., 1995, 38, 1247-1252; and U.S. Pat. No.5,602,273). However, it is also known that the hydroxylamines are ableto convert back to the parent N-oxides in vivo where the latter can bereduced in the hypoxic and reductive environment of tumerous cells wherethe underlying nitrogen mustards exerts their cytoxicity.

Referring to Scheme 10, in certain embodiments of compounds of Formula(AF), Formula (AG), and of Formula (AH) R¹ and/or R⁵, R⁶, R⁹, and thelinker L are defined as described herein; one of R², R³, and R⁴ is aN,N-bis-(2-functionalized)ethylamino group (nitrogen mustard group)linked to a connector group “A” (-A-N(CH₂—CH₂—R⁹)₂) wherein theconnector group “A” is a bond (“-”) or a methylene group (—CH₂—); eachof the remaining R², R³, and R⁴ is hydrogen; and each of R⁷ and R⁸ ishydrogen.

Referring to Scheme 10, N-oxidation of the N-mustard group of compoundsof Formula (AF) with a slight excess of 3-chloroperbezoic acid(meta-chloroperbenzoic acid, mCPBA) in a solvent such as dichloromethane(DCM) at about room temperature followed by work-up with aqueous sodiumhydrogencarbonate furnishes the more stable hydroxylamine (throughputative re-arrangement via a cyclic oxazetidinium species) of Formula(AG).

Referring to Scheme 10, N-oxidation of the N-mustard group of compoundsof Formula (AF) with 3-5 equivalents of peracetic acid (MeCO(O₂H)),prepared from 35 wt-% aqueous hydrogen peroxide (H₂O₂) in glacial aceticacid (HOAc), in a solvent such as dichloromethane (DCM) at about roomtemperature followed by acid extraction furnishes the correspondingN-oxide of Formula (AH).

Characterization

To determine the extent to which compounds provided by the presentdisclosure enter cells via the LAT1/4F2hc transporter, amino acid uptakeassays into cells that are transfected with DNA encoding the LAT1 and4F2hc subunits may be performed using, for example, HEK (human embryonickidney) or CHO (Chinese hamster ovary) cells. Oocytes may also beinjected with cRNA LAT1 and 4F2hc to express LAT1/4F2hc transporter.Compounds may be screened either for specificity for the LAT1/4F2hctransporter or for transport into cells endogenously expressing aplurality of transporters. The results of a screening method (e.g., acompetition uptake, exchange or direct uptake assay) using a cellexpressing the LAT1/4F2hc transporter may be compared with the resultsof a control cell(s) lacking the LAT1/4F2hc transporter or in thepresence of a specific inhibitor of the LAT1/4F2hc transporter.

In competition experiments, the ability of a compound to specificallybind to the LAT1/4F2hc transporter is determined. A known substrate(reference substrate) for the LAT1/4F2hc transporter and a test compoundare added to cells expressing the LAT1/4F2hc transporter. For example,gabapentin may be used as a reference because it demonstrates highselectivity for LAT1/4F2hc. Gabapentin is not a substrate for theintestinal amino acid transporters B^(0,+), ATB⁰⁺, and LAT2, whereasgabapentin may be a substrate for the organic cation transporter OCTN2(Cundy, et al., J Pharm Exp Ther, 2004, 311(1), 315-323; and Grigat, etal., Drug Metabol Disp, 2009, 37(2), 330-337). The amount or rate oftransport of the reference substrate in the presence of the testcompound is compared to the amount or rate of transport of the referencesubstrate in the absence of the test compound. If the amount or rate oftransport of the reference substrate is decreased by the presence of thetest compound, the test compound binds to the LAT1/4F2hc transporter.

Compounds that bind the LAT1/4F2hc transporter can be further analyzedto determine if they are transported by the LAT1/4F2hc transporter oronly compete for binding to the transporter. Transport of a compoundinto a cell can be determined by detecting a signal from within a cellfrom any of a variety of reporters. The reporter can be as simple as alabel such as a fluorophore, a chromophore, a radionuclide, or areporter can be an agent that is detected utilizing liquidchromatography-mass spectroscopy (LC/MS/MS). The same methods ofdetection can be used to determine if a reporter is transported from theintracellular space to the medium by administering the test compound tothe outside of the cell and sampling the media for the presence of theintracellular reporter after a predetermined period of time (exchangeassays).

Having determined that a compound is a substrate for LAT1/4F2hc, afurther screen may be performed to determine the selectivity of thecompound toward other membrane transporters. Selectivity refers to theaffinities with which a compound is transported by differenttransporters. In order to demonstrate selectivity for LAT1/4F2hc, acompound may be tested in uptake and/or competition assays for othertransporters. Transporters that could potentially transport LAT1/4F2hcsubstrates include SLC1A4 (ASCT1; NP_003029), SLC1A5 (ASCT2; NP_005619),SLC6A1 (GAT1; NP_003033), SLC6A5 (GlyT2; NP_004202), SLC6A6 (TauT;NP_003034), SLC6A8 (CT1; NP_005620), SLC6A9 (GlyT1; NM_008865), SLC6A11(GAT3; NP_55044), SLC6A12 (BGT1; NP_003035), SLC6A13 (GAT2; NP_057699),SLC6A14 (ATB^(0,+); NP_009162), SLC6A15 (B⁰AT2; NP_001139807), SLC6A17(XT1; NP_001010898), SLC6A18 (B⁰AT3; NP_872438), SLC6A19 (B⁰AT1;NP_001003841), SLC7A6 (y⁺LAT2; NP_001070253), SLC7A7 (y⁺LAT1;NP_001119577), SLC7A8 (LAT2; NP_036376), SLC7A9 (b^(0,+)AT; NP_055085),SCL7A10 (ASC-1; NP_062823), SLC15A1(PepT1; NP_005064), SLC15A2 (PepT2;NP_066568), SLC16A1 (MCT1; NP_003042), SLC16A2 (MCT8; NP_006508),SLC16A10 (TAT1; NP_061063), SLCO1B1 (OATP1B1; NP_006437), SLCO1B3(OATP1B3; NP_062818), SLC22A1 (OCT1; NP_003048), SLC22A2 (OCT2;NP_003049), SLC22A4 (OCTN1; NP_003050), SLC22A5 (OCTN2; NP_003051),SLC22A8 (OAT3; NP_004245), SLC36A1 (PAT1; NP_510968), SLC36A1 (PAT1;NP_510968), SLC36A2 (PAT2; NP_861441), SLC38A1 (SNAT1; NP_109599),SLC38A2 (SNAT2; NP_061849), SLC38A3 (SNAT3; NP_006832), SLC38A4 (SNAT4;NP_060488), SLC38A5 (SNAT5; NP_0277053), SLC43A1 (LAT3; NP_003618), andSLC43A2 (LAT4; NP_689559).

Human genes required for functional expression of a transporter ofinterest may be cloned using PCR, fully sequenced, and subcloned intoplasmids that can be used for expression in mammalian cells or Xenopuslaevis oocytes. Unless otherwise noted, all subunits of a transporter ofinterest are co-expressed in each heterologous system described in theexamples. Because many mammalian cell lines exhibit high levels of aminoacid transport activity, expression in Xenopus laevis oocytes can beadvantageous due to the low levels of endogenous amino acid transport.To assess transport function of a specific transporter protein, it canbe desirable to clone the cDNA and express the protein in cells thathave low endogenous transport activity. Competition assays may beperformed with labeled compounds that are optimal substrates (referencesubstrates) for the transporter of interest. Typically, uptake levels ofa test compound are compared to uptake of a reference substrate for thetransporter of interest.

Compounds of Formula (1) are substrates for LAT1/4F2hc and have aV_(max) of at least 10%, 20%, and in certain embodiments, at least 50%that of gabapentin. Concomitantly, the compounds have a low affinitytoward amino acid transporters of system A, system N, system ASC, andthe system L transporter LAT2/4F2hc.

Biodistribution studies with normal and tumor-bearing rats may be usedto determine the disposition of actively transported compounds and theselectivity of substrate accumulation in tissue that expresses theLAT1/4F2hc transporter compared with other tissue. Imaging techniquescan qualitatively and quantitatively elucidate the role of transportproteins in drug disposition, for example, whole body autoradiography(WBA). WBA allows both the visualization and the quantification ofradionuclide-labeled compound levels in a thin section of the wholeanimal. Information obtained using WBA is analogous to data obtainedfrom diagnostic imaging, albeit at a single point in time.

Pharmaceutical Compositions

Compounds of Formula (1) or pharmaceutically acceptable salts thereofmay be incorporated into pharmaceutical compositions to be administeredto a patient by any appropriate route of administration includingintradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, oral, peroral, sublingual, intracerebral,intravaginal, transdermal, rectal, inhalation, or topical. In certainembodiments, pharmaceutical compositions provided by the presentdisclosure are injectable formulations. In certain embodiments,pharmaceutical compositions provided by the present disclosure areinjectable intravenous formulations. In certain embodiments,pharmaceutical compositions provided by the present disclosure are oralformulations. Oral formulations may be oral dosage forms.

Pharmaceutical compositions provided by the present disclosure maycomprise a therapeutically-effective amount of a compound of Formula (1)or a pharmaceutically acceptable salt thereof together with a suitableamount of one or more pharmaceutically acceptable vehicles so as toprovide a composition for proper administration to a patient. Suitablepharmaceutical vehicles and methods of preparing pharmaceuticalcompositions are described in the art.

In certain embodiments, a compound of Formula (1) or a pharmaceuticallyacceptable salt thereof may be administered by intravenous injection.Suitable forms for injection include sterile aqueous solutions ordispersions of a compound of Formula (1). In certain embodiments, acompound may be formulated in a physiological buffer solution. Prior toadministration, a compound of Formula (1) or a pharmaceuticallyacceptable salt thereof may be sterilized by any art recognized thetechnique, including addition of antibacterial or antifungal agents, forexample, paraben, chlorobutanol, phenol, sorbic acid, thimersol, and thelike. In certain embodiments, a compound of Formula (1) or apharmaceutically acceptable salt thereof may be sterilized by filtrationbefore administration to a subject thereby minimizing or eliminating theneed for additional sterilization agents. An injectable dosage of acompound of Formula (1) may include from about 0.01 mL to about 10 mL,from about 0.1 mL to about 10 mL, from about 0.1 mL to about 5 mL, andin certain embodiments, from about 1 mL to about 5 mL.

Pharmaceutical compositions may comprise a therapeutically effectiveamount of one or more compounds of Formula (1), preferably in purifiedform, together with a suitable amount of a pharmaceutically acceptablevehicle, so as to provide a form for proper administration to a patient.When administered to a patient, the compounds and pharmaceuticallyacceptable vehicles are preferably sterile. Water is a preferred vehiclewhen the compound is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions may also be employed as liquidvehicles, particularly for injectable solutions. Suitable pharmaceuticalvehicles also include excipients such as starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Pharmaceuticalcompositions may also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. In addition, auxiliary, stabilizing,thickening, lubricating and coloring agents may be used.

Pharmaceutical compositions comprising a compound may be manufactured bymeans of conventional mixing, dissolving, granulating, levitating,emulsifying, encapsulating, entrapping or lyophilizing processes.Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers, diluents;excipients or auxiliaries, which facilitate processing of compounds intopreparations, which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

Pharmaceutical compositions provided by the present disclosure may takethe form of solutions, suspensions, emulsion, or any other form suitablefor use. Examples of suitable pharmaceutical vehicles are described inthe art.

For parenteral administration, compounds of Formula (1) may beincorporated into a solution or suspension. Parenteral administrationrefers to the administration by injection, for instance by intravenous,intracapsular, intrathecal, intrapleural, intratumoral, orintraperitoneal injection or intravesically. In certain embodiments, acompound of Formula (1) is administered intravenously.

A solution or suspension may also comprise at least one of the followingadjuvants: sterile diluents such as water for injection, saline, fixedoils, polyethylene glycols, glycerol, propylene glycol or othersynthetic solvents, antioxidants such as ascorbic acid or sodiumbisulfite, buffers such as acetates, citrates or phosphates, and agentsfor adjustment of the tonicity such as sodium chloride or dextrose. Aparenteral preparation may be enclosed into ampoules, disposablesyringes or multiple dosage vessels made of glass or plastic.

For topical administration, a compound of Formula (1) may be formulatedas a solution, gel, ointment, cream, suspension, etc. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedmay be used in the formulation. Such penetrants are generally known inthe art. Systemic formulations include those designed for administrationby injection, e.g., subcutaneous, intravenous, intramuscular,intrathecal or intraperitoneal injection, as well as those designed fortransdermal, transmucosal, oral or pulmonary administration. Systemicformulations may be made in combination with a further active agent thatimproves mucociliary clearance of airway mucus or reduces mucousviscosity. These active agents include, for example, sodium channelblockers, antibiotics, N-acetyl cysteine, homocysteine, sodium2-mercaptoethane sulfonate (MESNA), and phospholipids.

When a compound is acidic or basic it may be included in any of theabove-described formulations as the free acid or free base, apharmaceutically acceptable salt, a solvate of any of the foregoing, ora hydrate of any of the foregoing. Pharmaceutically acceptable saltssubstantially retain the activity of the free acid or base, may beprepared by reaction with bases or acids, and tend to be more soluble inaqueous and other protic solvents than the corresponding free acid orbase form.

Assessing single patient response to therapy and qualifying a patientfor optimal therapy are among the greatest challenges of modernhealthcare and relate to trends in personalized medicine. The novelβ-substituted γ-amino acid derivatives and β-substituted γ-amino acidanalogs provided by the present disclosure have a high selectivity forLAT1/4F2hc. Radio-labeled compounds for positron emission tomography(PET) or Single Photon Emission Computed Tomography (SPECT) with thesame selectivity toward LAT1/4F2hc may be used to predict the efficacyof the treatment based on a single-study, case-by-case patient analysisthus excluding subjects that are expected not to benefit from treatment.PET/SPECT scans using radiolabeled LAT1/4F2hc selective substrates, oncecorrelated to the concentration β-substituted γ-amino acid derivativesor β-substituted γ-amino acid analogs of Formula (1) can provide athree-dimensional distribution map, which can then be used formacroscopic dose calculations.

Accordingly, it is within the capability of those of skill in the art toassay and use the compounds of Formula (1) and/or pharmaceuticalcompositions thereof for therapy.

Therapeutic Dose

A compound of Formula (1) and/or pharmaceutical composition thereof willgenerally be used in an amount effective to achieve the intendedpurpose. For use to treat a disease such as cancer, a compound ofFormula (1) and/or pharmaceutical compositions thereof, may beadministered or applied in a therapeutically effective amount.

The amount of a compound of Formula (1) and/or pharmaceuticalcomposition thereof that will be effective in the treatment of aparticular disorder or condition disclosed herein will depend in part onthe nature of the disorder or condition, and can be determined bystandard clinical techniques known in the art. In addition, in vitro orin vivo assays may optionally be employed to help identify optimaldosage ranges. The amount of a compound of Formula (1) and/orpharmaceutical composition thereof administered will depend on, amongother factors, the subject being treated, the weight of the subject, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

A compound of Formula (1) may be assayed in vitro and in vivo, for thedesired therapeutic activity, prior to use in humans. For example, invitro assays may be used to determine whether administration of aspecific compound or a combination of compounds is preferred. Thecompounds may also be demonstrated to be effective and safe using animalmodel systems.

In certain embodiments, a therapeutically effective dose of a compoundof Formula (1) and/or pharmaceutical composition thereof will providetherapeutic benefit without causing substantial toxicity. Toxicity ofcompounds of Formula (1) and/or pharmaceutical compositions thereof maybe determined using standard pharmaceutical procedures and may bereadily ascertained by the skilled artisan. The dose ratio between toxicand therapeutic effect is the therapeutic index. In certain embodiments,a compound of Formula (1) and/or pharmaceutical composition thereofexhibits a particularly high therapeutic index in treating disease anddisorders. In certain embodiments, a dose of a compound of Formula (1)and/or pharmaceutical composition thereof will be within a range ofcirculating concentrations that include an effective dose with minimaltoxicity.

Kits

A compound of Formula (1), a pharmaceutically acceptable salt thereof,or a pharmaceutical composition of any of the foregoing may be includedin a kit that may be used to administer the compound to a patient fortherapeutic purposes. A kit may include a pharmaceutical compositioncomprising a compound of Formula (1) suitable for administration to apatient and instructions for administering the pharmaceuticalcomposition to the patient. In certain embodiments, a kit for use intreating cancer in a patient comprises a compound of Formula (1) or apharmaceutically acceptable salt thereof, a pharmaceutically acceptablevehicle for administering the compound, and instructions foradministering the compound to a patient.

Instructions supplied with a kit may be printed and/or supplied, forexample, as an electronic-readable medium, a video cassette, anaudiotape, a flash memory device, or may be published on an internet website or distributed to a patient and/or health care provider as anelectronic communication.

Therapeutic Uses

Compounds of Formula (1) may be used for treating cancer in a patient,wherein the cancerous tissue expresses the LAT1/4F2hc. In certainembodiments, the cancerous tissue expressing the LAT1/4F2hc transporteris in the brain of the patient.

Compounds of Formula (1) may be used in the treatment of a wide varietyof neoplasms where elevated LAT1/4F2hc mediated uptake occurs. Compoundsof Formula (1) are particularly useful for treating brain tumors,including metastases of other solid tumors, such as lung or breastcancer, in the brain.

In certain embodiments, a compound of Formula (1) or a pharmaceuticalcomposition comprising a compound of Formula (1) may be administered totreat a cancer known to be treated by an alkylating agent, such as, forexample, melphalan.

In certain embodiments, a compound of Formula (1) or a pharmaceuticalcomposition comprising a compound of Formula (1) may be used to treat,for example, one or more of the following cancers: adult and childhoodacute lymphoblastic leukemia (ALL), adult and childhood acute myeloidleukemia (AML), childhood adrenocortical carcinoma, a IDs-relatedcancers, a IDs-related lymphoma, anal cancer, appendix cancer,astrocytoma, childhood atypical teratoid/rhabdoid tumor, basal cellcarcinoma (nonmelanoma), extrahepatic bile duct cancer, childhoodbladder cancer, bone cancer, osteosarcoma, malignant fibroushistiocytoma, childhood craniopharyngioma, childhood brain stem glioma,adult and child brain tumor, childhood central nervous system embryonaltumors, childhood cerebellar astrocytoma, brain tumor, cerebralastrocytoma/malignant glioma, ductal carcinoma in situ, childhoodependymoblastoma, childhood ependymoma, childhood esthesioneuroblastoma,childhood medulloblastoma, childhood medulloepithelioma, childhoodpineal parenchymal tumors of intermediate differentiation,supratentorial primitive neuroectodermal tumors and pineoblastoma,childhood visual pathway and hypothalamic glioma, childhood brain andspinal cord tumors, adult and childhood breast cancer, male breastcancer, childhood bronchial tumors, hematopoetic tumors of the lymphoidlineage, hematopoetic tumors of the myeloid lineage, burkitt lymphoma,childhood carcinoid tumor, gastrointestinal carcinoid tumor, carcinomaof head and neck, childhood central nervous system embryonal tumors,primary central nervous system lymphoma, childhood cerebellarastrocytoma, cerebral astrocytoma/malignant glioma, childhood cervicalcancer, childhood cancers, childhood chordoma, chronic lymphocyticleukemia (CLL), chronic myeloproliferative disorders, colorectal cancer,cutaneous t-cell lymphoma, childhood central nervous system embryonaltumors, desmoplastic small round cell tumor, endometrial cancer,childhood ependymoblastoma, childhood ependymoma, esophageal cancer,childhood esophageal cancer, ewing family of tumors, childhoodextracranial germ cell tumor, extragonadal germ cell tumor, extrahepaticbile duct cancer, dye cancer, Intraocular melanoma, retinoblastoma,gallbladder cancer, gastric (stomach) cancer, childhood gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (gIst), childhood gastrointestinal stromal cell tumor,childhood extracranial germ cell tumor, extragonadal germ cell tumor,ovarian germ cell tumor, gestational trophoblastic tumor/disease, adultglioma, glioblastoma, childhood brain stem, childhood cerebralastrocytoma, childhood visual pathway and hypothalamic glioma, hairycell leukemia, childhood heart cancer, head and neck cancer, childhoodhead and neck cancer, adult (primary) hepatocellular (liver) cancer,childhood (primary) hepatocellular (liver) cancer, adult Hodgkinlymphoma, childhood Hodgkin lymphoma, hypopharyngeal cancer, childhoodhypothalamic and visual pathway glioma, intraocular melanoma, pancreaticneuroendocrine tumors (islet cell tumors), endocrine pancreas tumors(islet cell tumors), Kaposi sarcoma, kidney (renal cell) cancer, kidneycancer, laryngeal cancer, childhood laryngeal cancer, adult acutelymphoblastic leukemia, childhood acute lymphoblastic leukemia, adultacute myeloid leukemia, childhood acute myeloid leukemia, chronicmyelogenous leukemia (cml), hairy cell leukemia, lip and oral cavitycancer, adult primary liver cancer, childhood primary liver cancer,non-small cell lung cancer, small cell lung cancer, a IDs-relatedlymphoma, Burkitt lymphoma, t-cell lymphoma, b-cell lymphoma, cutaneoust-cell lymphoma, adult Hodgkin lymphoma, childhood Hodgkin lymphoma,adult non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, primarycentral nervous system lymphoma, langerhans cell histiocytosis,Waldenstrom macroglobulinemia, malignant fibrous histiocytoma of boneand osteosarcoma, childhood medulloblastoma, childhoodmedulloepithelioma, melanoma, intraocular (dye) melanoma, Merkel cellcarcinoma, adult malignant mesothelioma, childhood mesothelioma, primarymetastatic squamous neck cancer with occult, mouth cancer,myelodysplastic/myeloproliferative neoplasms, midline tract carcinomainvolving nUt gene, childhood multiple endocrine neoplasia syndrome,multiple myeloma/plasma cell neoplasm, mycosis fungoides,myelodysplastic syndromes myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, adult acute myeloid leukemia, childhoodacute myeloid leukemia, multiple myeloma, chronic myeloproliferativedisorders, malignant germ cell tumors, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, childhood nasopharyngeal cancer,neuroblastoma, adult non-Hodgkin lymphoma, childhood non-Hodgkinlymphoma, non-small cell lung cancer, childhood oral cancer, lip andoral cavity cancer, oropharyngeal cancer, osteosarcoma and malignantfibrous histiocytoma of bone, childhood ovarian cancer, ovarianepithelial cancer, ovarian germ cell tumor, ovarian low malignantpotential tumor, pancreatic cancer, childhood pancreatic cancer, isletcell tumors, childhood papillomatosis, paranasal sinus and nasal cavitycancer, parathyroid cancer, penile cancer, pharyngeal cancer,pheochromocytoma, childhood pineal parenchymal tumors of intermediatedifferentiation, childhood pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, paraganglioma, plasma cellneoplasm/multiple myeloma, pleuropulmonary blastoma, childhoodpleuropulmonary blastoma, primary central nervous system (cns) lymphoma,pregnancy and breast cancer, primary central nervous system lymphoma,prostate cancer, rectal cancer, renal cell (kidney) cancer, childhoodrenal cell (kidney) cancer, renal pelvis and ureter, transitional cellcancer, respiratory tract carcinoma involving the nUt gene on chromosome15, retinoblastoma, childhood rhabdomyosarcoma, salivary gland cancer,childhood salivary gland cancer, sarcoma (dwing family of tumors),Kaposi sarcoma, adult soft tissue sarcoma, childhood soft tissuesarcoma, uterine sarcoma, sezary syndrome, skin cancer (nonmelanoma),childhood skin cancer, melanoma, Merkel cell skin carcinoma, small celllung cancer, small intestine cancer, adult soft tissue sarcoma,childhood soft tissue sarcoma, squamous cell carcinoma (nonmelanoma),primary and metastatic squamous neck cancer with occult, stomach(gastric) cancer, childhood stomach (gastric) cancer, childhoodsupratentorial primitive neuroectodermal tumors, cutaneous t-celllymphoma, testicular cancer, throat cancer, thymoma and thymiccarcinoma, childhood thymoma and thymic carcinoma, thyroid cancer,childhood thyroid cancer, gestational trophoblastic tumor, adult unknownprimary site, carcinoma of, childhood cancer of unknown primary site,unusual cancers of childhood, transitional cell cancer of ureter andrenal pelvis, urethral cancer, endometrial uterine cancer, uterinesarcoma, vaginal cancer, childhood vaginal cancer, childhood visualpathway and hypothalamic glioma, vulvar cancer, Waldenstrommacroglobulinemia, Wilms tumor, and women's cancers.

In certain embodiments, a compound of Formula (1) or a pharmaceuticalcomposition comprising a compound of Formula (1) may be used to treat,for example, one or more of the following cancers wherein the cancer isselected from any of the primary adult and childhood brain and CNScancers including glioblastoma (GBM) and astrocystoma, skin cancersincluding melanoma, lung cancers including small cell lung cancers,non-small cell lung cancers (NSCLC), and large cell lung cancers,breasts cancers including triple negative breast cancer (TNBC), bloodcancers including myelodysplastic syndrome (MDS), multiple myeloma (MM),and acute myeloid leukemia (AML), prostate cancer including castrateresistant prostate cancer (CRPC), liver cancers including hepatocellularcarcinoma (HCC), esophageal and gastric cancers, and any systemic andcentral metastases of any of the foregoing.

Compounds of Formula (1) maybe used to treat a cancer in which there isdifferential LAT1/4F2hc transport activity relative to surroundingtissue and/or tissue in other body organs. Patients having a tumorexhibiting a greater LAT1/4F2hc transport activity than non-diseasedtissue are expected to respond more favorably to treatment with atherapeutic agent that is a substrate for the LAT1/4F2hc transporter andto experience fewer adverse effects associated with the effects of thetherapeutic agent on non-diseased tissue. Compounds of Formula (1) aretherapeutic agents, are substrates for the LAT1/4F2hc transporter, andexhibit cytotoxicity.

The amount of a compound of Formula (1) that will be effective in thetreatment of a cancer will depend, at least in part, on the nature ofthe disease, and may be determined by standard clinical techniques knownin the art. In addition, in vitro or in vivo assays may be employed tohelp identify optimal dosing ranges. Dosing regimens and dosingintervals may also be determined by methods known to those skilled inthe art. The amount of compound of Formula (1) administered may dependon, among other factors, the subject being treated, the weight of thesubject, the severity of the disease, the route of administration, andthe judgment of the prescribing physician.

For systemic administration, a therapeutically effective dose may beestimated initially from in vitro assays. Initial doses may also beestimated from in vivo data, e.g., animal models, using techniques thatare known in the art. Such information may be used to more accuratelydetermine useful doses in humans. One having ordinary skill in the artmay optimize administration to humans based on animal data.

A dose of compound of Formula (1) and appropriate dosing intervals maybe selected to maintain a sustained therapeutically effectiveconcentration of the compound of Formula (1) in the blood of a patient,and in certain embodiments, without exceeding a minimum adverseconcentration.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered once per day, twice per day,and in certain embodiments at intervals of more than once per day.Dosing may be provided alone or in combination with other drugs and maycontinue as long as required for effective treatment of the disease.Dosing may also be undertaken using continuous or semi-continuousadministration over a period of time. Dosing includes administering apharmaceutical composition to a mammal, such as a human, in a fed orfasted state.

A pharmaceutical composition may be administered in a single dosage formor in multiple dosage forms or as a continuous or an accumulated doseover a period of time. When multiple dosage forms are used the amount ofcompound of Formula (1) contained within each of the multiple dosageforms may be the same or different.

Suitable daily dosage ranges for administration may range from about 2mg to about 50 mg of a compound of Formula (1) per kilogram body weight.

Suitable daily dosage ranges for administration may range from about 1mg to about 100 mg of a compound of Formula (1) per square meter (m²) ofbody surface.

In certain embodiments, a compound of Formula (1) may be administered totreat cancer in a subject in an amount from about 50 mg to about 2,000mg per day, from about 100 mg to about 1,500 mg per day, from about 200mg to about 1,000 mg per day, or in any other appropriate daily dose.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered to treat cancer in a subjectso as to provide a therapeutically effective concentration of a compoundof Formula (1) in the blood or plasma of the subject. In certainembodiments, a therapeutically effective concentration of a compound ofFormula (1) in the blood or plasma of a subject is from about 1 μg/mL toabout 60 μg/mL, from about 2 μg/mL to about 50 μg/mL, from about 5 μg/mLto about 40 μg/mL, from about 5 μg/mL to about 20 μg/mL, and in certainembodiments, from about 5 μg/mL to about 10 μg/mL. In certainembodiments, a therapeutically effective concentration of a compound ofFormula (1) in the blood or plasma of a subject is at least about 2μg/mL, at least about 5 μg/mL, at least about 10 μg/mL, at least about15 μg/mL, at least about 25 μg/mL, and in certain embodiments, at leastabout 30 μg/mL. In certain embodiments, a therapeutically effectiveconcentration of a compound of Formula (1) in the blood or plasma of asubject is less than an amount that causes unacceptable adverse effectsincluding adverse effects to homeostasis. In certain embodiments, atherapeutically effective concentration of a compound of Formula (1) inthe blood or plasma of a subject is an amount sufficient to restoreand/or maintain homeostasis in the subject.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered to treat cancer in a subjectso as to provide a therapeutically effective concentration of a compoundof Formula (1) in the blood or plasma of a subject for an extendedperiod of time such as, for example, for at least about 4 hours, for atleast about 6 hours, for at least about 8 hours, for at least about 10hours, and in certain embodiments, for at least about 12 hours.

The amount of a compound of Formula (1) administered may vary during atreatment regimen.

Pharmaceutical compositions provided by the present disclosure mayfurther comprise one or more pharmaceutically active compounds inaddition to a compound of Formula (1). Such compounds may be provided totreat the cancer being treated with the compound of Formula (1) or totreat a disease, disorder, or condition other than the cancer beingtreated with the compound of Formula (1).

In certain embodiments, a compound of Formula (1) may be used incombination with at least one other therapeutic agent. In certainembodiments, a compound of Formula (1) may be administered to a patienttogether with another compound for treating cancer in the subject. Incertain embodiments, the at least one other therapeutic agent may be adifferent compound of Formula (1). A compound of Formula (1) and the atleast one other therapeutic agent may act additively or, and in certainembodiments, synergistically. The at least one additional therapeuticagent may be included in the same pharmaceutical composition or vehiclecomprising the compound of Formula (1) or may be in a separatepharmaceutical composition or vehicle. Accordingly, methods provided bythe present disclosure further include, in addition to administering acompound of Formula (1), administering one or more therapeutic agentseffective for treating cancer or a different disease, disorder orcondition than cancer. Methods provided by the present disclosureinclude administration of a compound of Formula (1) and one or moreother therapeutic agents provided that the combined administration doesnot inhibit the therapeutic efficacy of a compound of Formula (1) and/ordoes not produce adverse combination effects.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered concurrently with theadministration of another therapeutic agent, which may be part of thesame pharmaceutical composition as, or in a different pharmaceuticalcomposition than that comprising a compound of Formula (1). A compoundof Formula (1) may be administered prior or subsequent to administrationof another therapeutic agent. In certain embodiments of combinationtherapy, the combination therapy may comprise alternating betweenadministering a compound of Formula (1) and a composition comprisinganother therapeutic agent, e.g., to minimize adverse drug effectsassociated with a particular drug. When a compound of Formula (1) isadministered concurrently with another therapeutic agent thatpotentially may produce an adverse drug effect including, for example,toxicity, the other therapeutic agent may be administered at a dose thatfalls below the threshold at which the adverse drug reaction iselicited.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered with one or more substancesto enhance, modulate and/or control release, bioavailability,therapeutic efficacy, therapeutic potency, stability, and the like of acompound of Formula (1). For example, to enhance the therapeuticefficacy of a compound of Formula (1), a compound of Formula (1) or apharmaceutical composition comprising a compound of Formula (1) may beco-administered with one or more active agents to increase theabsorption or diffusion of the compound of Formula (1) from thegastrointestinal tract to the systemic circulation, or to inhibitdegradation of the compound of Formula (1) in the blood of a subject. Incertain embodiments, a pharmaceutical composition comprising a compoundof Formula (1) may be co-administered with an active agent havingpharmacological effects that enhance the therapeutic efficacy of thecompound of Formula (1).

In certain embodiments, a compound of Formula (1) or a pharmaceuticalcomposition comprising a compound of Formula (1) may be administered inconjunction with an agent known or believed to be effective in treatingcancer in a patient.

For example, in certain embodiments, a compound of Formula (1) or apharmaceutical composition comprising a compound of Formula (1) may beadministered in conjunction with another chemotherapeutic agents, suchas, for example, N-acetyl cysteine (NAC), adriamycin, alemtuzumab,amifostine, arsenic trioxide, ascorbic acid, bendamustine, bevacizumab,bortezomib, busulfan, buthionine sulfoxime, carfilzomib, carmustine,clofarabine, cyclophosphamide, cyclosporine, cytarabine, dasatinib,datinomycin, defibrotide, dexamethasone, docetaxel, doxorubicin,etoposide, filgrastim, floxuridine, fludarabine, gemcitabine, interferonalpha, ipilimumab, lenalidomide, leucovorin, melphalan, mycofenolatemofetil, paclitaxel, palifermin, panobinostat, pegfilrastim,prednisolone, prednisone, revlimid, rituximab, sirolimus, sodium2-mercaptoethane sulfonate (MESNA), sodium thiosulfate, tacrolimus,temozolomide, thalidomide, thioguanine, thiotepa, topotecan, velcade, ora combination of any of the foregoing. In certain embodiments, acompound of Formula (1) and/or pharmaceutical compositions thereof canbe used in combination therapy with other chemotherapeutic agentsincluding one or more antimetabolites such as folic acid analogs;pyrimidine analogs such as fluorouracil, floxuridine, and cytosinearabinoside; purine analogs such as mercaptopurine, thiogunaine, andpentostatin; natural products such as vinblastine, vincristine,etoposide, tertiposide, dactinomycin, daunorubicin, doxurubicin,bleomycin, mithamycin, mitomycin C, L-asparaginase, and interferonalpha; platinum coordination complexes such as cis-platinum, andcarboplatin; mitoxantrone; hydroxyurea; procarbazine; hormones andantagonists such as prednisone, hydroxyprogesterone caproate,medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol,ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone,flutamide, and leuprolide, anti-angiogenesis agents or inhibitors suchas angiostatin, retinoic acids, paclitaxel, estradiol derivatives, andthiazolopyrimidine derivatives; apoptosis prevention agents; andradiation therapy.

In certain embodiments, a compound of Formula (1) may be coadministeredwith a compound that inhibits DNA repair such as, for example,O6-benzylguanine (O6-BG). In certain embodiments, a compound of Formula(1) may be coadministered with a compound that blocks and/or inhibitstransporters other than LAT1 such as, for example, amino acids. Incertain embodiments, compounds of Formula (1) may be administered to apatient together with one or more amino acids such as, for example,arginine (Arg), serine (Ser), lysine (Lys), asparagine (Asn), glutamine(Gln), threonine (Thr), or mixtures of any of the foregoing. In certainembodiments, co-aministration of amino acids is intended to saturateamino acid transporters that interact with compounds of Formula (1) andthereby increase the selectivity for LAT1.

The efficacy of administering a compound of Formula (1) for treatingcancer may be assessed using in vitro and animal studies and in clinicaltrials.

The suitability of compounds of Formula (1) and/or pharmaceuticalcompositions thereof in treating cancers listed above may be determinedby methods described in the art. For example, screens developed todemonstrate the anti-tumor activity of oncolytic agents are known(Miller, et al., J Med Chem, 1977, 20(3), 409-413; Sweeney, et al.,Cancer Res, 1978, 38(9), 2886-2891; and Weiss and Von Hoff, Semin Oncol,1985, 12(3 Suppl 4), 69-74). Accordingly, it is well with the capabilityof those of skill in the art to assay and use the compounds and/orpharmaceutical compositions thereof to treat the above diseases ordisorders.

Methods provided by the present disclosure have use in animals,including mammals, such as in humans.

General Experimental Protocols

All reagents and solvents were purchased from commercial suppliers andused without further purification or manipulation.

Proton NMR spectra were recorded on a Varian Mercury Plus300 MHzSpectrometer equipped with an Oxford magnet, a Sun Sunblade 150 hostcomputer, a Solaris operating system, VNMR data processing software, anda HP LaserJet printer. CDCl₃ (99.8% D), MeOH-d⁴ (CD₃OD, 99.8+% D),deuteroxide (D₂O) (99.8+% D) were used as recording solvents unlessotherwise noted. The CHCl₃, MeOH-d³, HDO solvent signals ortetramethylsilane (TMS) were used for calibration of the individualspectra.

Analytical thin layer chromatography (TLC) was performed using EMDMillipore aluminum-backed TLC sheets (EMD5554-7) pre-coated with silicagel 60 F254 (200 μm thickness, 60 Å pore size) where F254 is afluorescent indicator with a 254 nm excitation wavelength. An ENF-240CSpectroline® UV-lamp (Spectronics Corporation, USA) was used for TLCdetection and visualization. Dyeing or staining reagents for TLCdetection and visualization, e.g., an ethanolic ninhydrin solution or a0.2 wt-% aqueous potassium permanganate (KMnO₄) solution, were preparedaccording methods known in the art.

Analytical LC/MS was performed on a Shimadzu LC/MS-2020 ProminenceSeries system equipped with CBM-20A communication bus module (Shimadzu228-45012-32), a SPD-20AV UV/VIS detector (Shimadzu 228-45004-32), aSIL-20AC autosampler (Shimadzu 228-45136-32), DGU-20A5 degasser(Shimadzu 228-45019-32), two LC-20AD XP HPLC pumps (Shimadzu228-45137-32), an Agilent Zorbax 5 μm XDB-C18 2.1×50 mm column (Agilent960 967-902), and a commercial desktop computer and printer for datacomputation. Gradients of water (solvent A) (Arrowhead, Nestle NorthAmerica, Inc.) and acetonitrile (MeCN; solvent B) (EMD AX0145-1 orAldrich CHROMASOLV® 439134) containing 0.075 vol-% of formic acid (EMDFX0440-7) were used in analytical LC/MS analyses.

Analytical LC/UV was performed on an Agilent 1100 Series system equippedwith an Agilent 1100 Series degasser (Agilent G1379A), an Agilent 1100Series quad pump (Agilent G1311A), an Agilent 1100 Series autosampler(ALS) (Agilent G1329A), an Agilent 1100 Series COLCOM (Agilent G1316A),a Phenomenex Gemini C18 5 μm 110 Å pore size 150×4.6 mm HPLC column(Phenomenex 00F-4435-E0), a Compaq Presario personal computer, and a HPLaserJet P2015 printer for data computation. Gradients of water (solventA) (Arrowhead, Nestle North America, Inc.) and acetonitrile (MeCN;solvent B) (EMD AX0145-1 or Aldrich CHROMASOLV® 439134) containing 0.075vol-% of formic acid (EMD FX0440-7) were used in analytical LC/UVanalyses.

Preparative HPLC was conducted with a Varian ProStar Series systemequipped with a Model 340 UV-C UV-VIS detector, a Model 210 solventdelivery module, a Hamilton PRP-112-20 μm 100 Å 21.2×250 mm preparativeHPLC column (Hamilton 79428), and a commercial desktop personal computerfor data computation. Gradients of water (solvent A) (Arrowhead, NestleNorth America, Inc.) and acetonitrile (MeCN; solvent B) (EMD AX0145-1 orAldrich CHROMASOLV® 439134) containing 0.1 vol-% of formic acid (EMDFX0440-7) were used for preparative HPLC purifications.

Compound isolation from aqueous solvent mixtures, e.g.,acetonitrile/water/0.1 vol-% formic acid, was accomplished by primarylyophilization of pooled and frozen (after freeze drying) fractionsunder reduced pressure at room temperature using manifold freeze dryerssuch as Heto Drywinner DW 6-85-1, Heto FD4, or VIRTIS Freezemobile 25 ESequipped with a high vacuum pump. Optionally, and if the isolatedcompound had ionizable functional groups such as an amino group or acarboxylic acid, the lyophilization process was conducted in thepresence of an excess (about 1.1 to 5.0 equivalents) of 1.0 Mhydrochloric acid (HCl) to yield the purified compound(s) as thecorresponding hydrochloride salt (HCl-salt), dihydrochloride salts,and/or the corresponding protonated free carboxylic acid. Melting pointswere determined in duplicate with a SRS OptiMelt MPA-100 automatedmelting point system with digital imaging processing technology and areuncorrected (Stanford Research Systems, USA).

Filtrations were conducted using commercial Celite® 545 (EMD CX0574-1)which was compressed in to glass Büchner-funnels to create a plug of 2-5cm thickness. Reaction mixtures containing precipitated reaction sideproducts or heterogenous catalyst residues were filtered off usingstandard techniques. Care must be taken filtering off activatedcatalysts or finely dispersed metals (ignition!).

Unless otherwise noted, aqueous work-up typically constitutes dilutionof a crude reaction product, with or without residual reaction solvent,with 1.0 M hydrochloric acid (HCl) or a saturated aqueous solution ofammonium chloride (NH₄Cl), multiple extraction with an organic solvent,e.g., ethyl acetate (EtOAc), diethyl ether (Et₂O), or dichloromethane(DCM), washing with water, a saturated aqueous solution of sodiumhydrogencarbonate (NaHCO₃), and brine (saturated aqueous solution ofsodium chloride (NaCl)), drying of the organic phase (combined organicextracts) over anhydrous magnesium sulfate (MgSO₄) (EMD MX0075-1) orsodium sulfate (Na₂SO₄) (EMD SX0760E-3), filtration, washing of thefilter residue, and evaporation of the combined filtrates under reducedpressure using a rotary evaporator at room or elevated temperaturefollowed by compound purification e.g., silica gel columnchromatography, crystallization or titruation.

Silica gel column chromatography was conducted with silica gel (about100-200 mL silica gel per gram of compound) 600.04-0.063 mm (40-63 μm,230-400 mesh) (EMD MilliporeEM1.09385.9026/EM1.09385.1033/EM1.09385.2503) using single solvents ormixtures of suitable solvents, e.g., ethyl acetate (EtOAc) and hexane ordichloromethane (DCM) and methanol (MeOH), as determined by TLC.Samples/fractions containing desired product detected by analytical TLCand/or analytical LC/MS, or LC/UV were pooled and the solvents wereremoved under reduced pressure using a Heidolph Laborota 4001 Efficientrotary evaporator (Heidolph, Germany) (Heidolph 519-10000-01-5) equippedwith a HB digit heating bath (Heidolph 517-01002-01-4), and a Rotavacvalve control vacuum pump (Heidolph 591-00130-01-0).

Chemical names were generated using the ChemDraw Ultra 12.0(CambridgeSoft, Cambridge, Mass., USA) nomenclature program.

Description 1 General Procedure for Aromatic Nitration

Variant A:

Adapting literature known protocols (Harmon, et al., U.S. Pat. No.5,959,113; International Application Publication No. WO 2007/021937;International Application Publication No. WO 2008/021369; U.S. PatentPublication No. 2008/0045534; International Application Publication No.WO 2005/110416; and Palmer, et al., J. Med. Chem., 1996, 39(13),2518-2528), a solution of the aromatic aldehyde or ketone (20 mmol) isdissolved in glacial acetic acid (35 mL). The solution is cooled toabout 0° C. (ice bath). The solvent may solidify. To the reactionmixture is added white fuming nitric acid (min. 90 wt-% HNO₃) (35-70 mL)using an addition funnel upon which the reaction mixture becomes liquidagain. The reaction mixture is stirred with slow warming to roomtemperature and followed by TLC and/LC/MS to completion. The reactionmixture is poured onto crushed ice (150-300 g). Upon complete melting ofthe ice, the aqueous phase is extracted with dichloromethane (DCM). Theaqueous phase is extracted with DCM (2×) and the combined organicextracts are successively washed with a saturated aqueous sodiumhydrogencarbonate (NaHCO₃) solution (2×), and brine (lx), dried overanhydrous magnesium sulfate (MgSO₄), filtered, and the solvents wereevaporated under reduced pressure using a rotary evaporator. The crudematerial is purified by silica gel column chromatography or isre-crystallized. Nitration regioisomers may also be separated by silicagel chromatography.

Variant B:

Adapting literature known protocols (Svenstrup, et al., ChemMedChem,2008, 3(10), 1604-1615), the aromatic aldehyde or ketone (20 mmol) isadded to concentrated sulfuric acid (conc. H₂SO₄) (6 mL) (exothermic!)while the temperature is maintained below 5° C. A cold mixture of conc.H₂SO₄ (3 mL) and white fuming nitric acid (min. 90 wt-% HNO₃) (d>1.5g/mL, 2 mL) is added dropwise (very exothermic!) whilst keeping thetemperature under 5° C. (acetone/solid CO₂ cooling bath). The reactionmixture is stirred at this temperature and followed by TLC and/LC/MS tocompletion. Work-up and product isolation and purification are conductedas described for Variant A.

Description 2 General Procedure for the Horner-Wadsworth-EmmonsOlefination

Variant A:

Adapting literature known protocols (Blanchette, et al., TetrahedronLett., 1984, 25, 2183-2186; Rathke and Novak, J. Org. Chem., 1985, 50,2624-2627; and Claridge, et al., Org. Lett., 2008, 10(23), 5437-5440), asolution of a trialkyl phosphonoacetate (20-25 mmol), triethylamine(Et₃N, TEA), diisopropylethylamine (DIPEA, Hinigs-base), or1,8-diazabicylco[5.4.0]undec-7-ene (DBU) (22-27.5 mmol), and anhydrouslithium bromide (LiBr), lithium chloride (LiCl), magnesium bromide(MgBr2) (24-30.0 mmol) in anhydrous acetonitrile (MeCN) ortetrahydrofuran (THF) (20-40 mL) is cooled to about 0° C. (ice bath).Solid aldehyde (20 mmol) or a solution of the aldehyde (20 mmol) in asmall amount of MeCN or THF is added in small portions. The reactionmixture is stirred with warming to room temperature for 1 to 12 hoursunder a nitrogen atmosphere. The reaction is monitored by TLC and/orLCMS to completion. The reaction is quenched by addition of water. Themajority of the volatiles (THF) may be evaporated under reduced pressure(rotary evaporator; ambient bath temperature) prior to further work-up.The residue is diluted with 1.0 M hydrochloric acid and extracted withethyl acetate (EtOAc). The aqueous phase is extracted with additionalEtOAc (2×). The combined organic extracts are successively washed with asaturated aqueous sodium hydrogencarbonate (NaHCO₃) solution (1×) andwith brine (1×), dried over anhydrous magnesium sulfate (MgSO₄),filtered, and the solvents are evaporated to dryness under reducedpressure. The crude material is purified by silica gel columnchromatography or is re-crystallized. Geometric isomers((E)/(Z)-isomeric mixtures) may also be separated by silica gelchromatography.

Variant B:

Adapting literature known protocols (U.S. Pat. No. 6,313,312; andBryans, et al., J. Med. Chem. 1998, 41, 1838-1845), a solution of atrialkyl phosphonoacetate (22-24 mmol) in anhydrousN,N-dimethylformamide (DMF) (80 mL) is cooled to about 0° C. (ice bath)under a nitrogen atmosphere. Sodium hydride (60 wt-% suspension of NaHin mineral oil) (22-25 mmol) is added in small portions (H₂-gasevolution and exotherm!) under a nitrogen blanket. The reaction mixtureis stirred at this temperature until gas evolution has ceased (about30-60 minutes). The aldehyde or ketone (20 mmol) is added either insolid form in small portions or dropwise as a solution in a small amountof anhydrous DMF. The reaction mixture is stirred with warming to roomtemperature for 1-12 hours under a nitrogen atmosphere. The reaction ismonitored by TLC and/or LCMS to completion. Work-up and productisolation and purification are conducted as described for Variant A.

Description 3 General Procedure for the 1,4-Conjugate Addition ofNitroalkane

Adapting literature known protocols (Altenbach, et al., J. Med. Chem.2004, 47, 3220-3235; Bryans, et al., J. Med. Chem. 1998, 41, 1838-1845;Marivet, et al., J. Med. Chem. 1989, 32, 1450-1457; Ono, et al.,Synthesis, 1984, 226-227; Bunce, et al., Org. Pre. Proc. Int., 1987,19(6), 471-475; Crosby, et al., Synlett, 2010, 539-542; Roberts, et al.,Bioorg. Med. Chem. Lett., 2009, 19(11), 3113-3117; and Whitlock, et al.,Bioorg. Med. Chem. Lett., 2009, 19(11), 3118-3121), the α,β-unsaturatedester (15 mmol) is dissolved in acetonitrile (MeCN) or tetrahydrofuran(THF) (15-20 mL) and nitroalkane (˜150 mmol). Depending on solubility,the α,β-unsaturated ester (15 mmol) may also be dissolved in neatnitroalkane (25 mL). The solution is cooled to about 0° C. (ice bath).To the cooled reaction mixture is drop-wise added neat1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (15-17 mmol). The reactionmixture is stirred overnight with gradual warming to room temperature orheated to 50-70° C. for 1-24 hours. The reaction is followed by TLCand/or LC/MS to completion. Alternatively, tetramethyl guanidine (TMG)(1-2 mmol) or 1.0 M tetrabutylammonium fluoride (Bu₄NF, TBAF) in THF)(15 mL, 15 mmol) can be used as a base. The reaction mixture is dilutedand acidified with 1.0 M hydrochloric acid (pH<2). The aqueous phase isextracted with ethyl acetate (EtOAc). The combined organic extracts aresuccessively washed with a saturated aqueous sodium hydrogencarbonate(NaHCO₃) solution (1×) and with brine (1×), dried over anhydrousmagnesium sulfate (MgSO₄), filtered, and the solvents are evaporated todryness under reduced pressure. The crude material is purified by silicagel column chromatography or is re-crystallized.

Description 4 General Procedure for the Raney-Nickel Catalyzed Reductionand Lactamization

Adapting literature known protocols (Altenbach, et al., J. Med. Chem.2004, 47, 3220-3235; Bryans, et al., J. Med. Chem. 1998, 41, 1838-1845;Marivet, et al., J. Med. Chem. 1989, 32, 1450-1457; Ono, et al.,Synthesis, 1984, 226-227; Bunce, et al., Org. Pre. Proc. Int., 1987,19(6), 471-475; Crosby, et al., Synlett, 2010, 539-542; Roberts, et al.,Bioorg. Med. Chem. Lett., 2009, 19(11), 3113-3117; and Whitlock, et al.,Bioorg. Med. Chem. Lett., 2009, 19(11), 3118-3121), a slurry of activeRaney®-3202 nickel (5-10 mL) is washed with distilled water (3×) (pH ofsupernatant ˜8-8.5) and 200-proof ethanol (EtOH) (3×). The catalyst istransferred with EtOH (2×10 mL) into a 500 mL Parr hydrogenation vesselcontaining a solution of the 4-nitrobutanoate (10 mmol) in EtOH (10-30mL). After three evacuation/refill cycles, the reaction mixture isshaken at room temperature for 4-24 hours at a hydrogen pressure ofabout 50-60 psi. The reaction is followed by TLC and/or LC/MS tocompletion. The supernatant is decanted off and the catalyst isthoroughly washed with methanol (MeOH) and decanted. The combinedorganic solutions are filtered over a short plug of Celite® 545 asfilter aid (about 2 cm) and the alcoholic solvents are evaporated underreduced pressure using a rotary evaporator. The ratio of cyclized(γ-lactamized) reaction product vs. non-cyclized reduction product isdetermined by ¹H NMR analysis (300 MHz, CDCl₃) and/or TLC and LC/MS. Tocomplete lactam formation of non-cyclized material, the crude materialis dissolved/suspended in a sealed tube in toluene and heated to about70-95° C. (oil bath temperature) for 1-12 h. Alternatively, the crudematerial is dissolved in MeOH and heated with slow evaporation of thesolvent for 1-6 h. After removal of the solvent under reduced pressure,the crude material is purified by silica gel column chromatography or isre-crystallized.

Description 5 General Procedure for the Reduction of Benzylic Ketones toBenzylic Alcohols

Adapting literature known protocols (Felder, et al., J. Med. Chem.,1970, 13(3), 559-561; Svenstrup, et al., ChemMedChem, 2008, 3(10),1604-1615; and Shen and Jensen, DE Patent No. 2731292A1 (1978)), thearomatic ketone (10 mmol) is dissolved in methanol (MeOH) or ethanol(EtOH) (20 mL). Sodium borohydride (NaBH₄) (5 mmol) is added in smallportions. The reaction mixture is stirred for about 1-12 h at about roomtemperature. The reaction is monitored by TLC and/or LCMS to completion.The reaction is quenched by addition of 1 M hydrochloric acid (HCl). Themajority of the volatiles are evaporated under reduced pressure (rotaryevaporator; ambient bath temperature). The residue is diluted with 1 Mhydrochloric acid and extracted with ethyl acetate (EtOAc). The aqueousphase is extracted with additional EtOAc (2×). The combined organicextracts are successively washed with a saturated aqueous sodiumhydrogencarbonate (NaHCO₃) solution (1×) and with brine (1×), dried overanhydrous magnesium sulfate (MgSO₄), filtered, and the solvents areevaporated to dryness under reduced pressure. If needed, the crudematerial is purified by silica gel column chromatography or isre-crystallized.

Description 6 General Procedure for the Reduction of Benzoic Acids toBenzylic Alcohols

Adapting literature known protocols (Hay, et al., J. Chem. Soc., PerkinTrans. 1, 1999, 2759-2770; Fujikawa, et al., J. Am. Chem. Soc., 2008,130, 14533-14543; International Publication No. WO 2010/122089; andInternational Application Publication No. WO2008/031594), commercialborane dimethylsulfide (BH₃.DMS, BH₃.SMe₂) (2.0 M in THF) (50 mL, 100mmol) or borane tetrahydrofurane complex (BH₃.THF) (1.0 M in THF) (100mL, 100 mmol) is added dropwise at room temperature to a stirredsolution of the nitrobenzoic acid (50 mmol) in anhydrous THF (250 mL).Optionally, the reaction is performed in the presence of trimethylborate (B(OMe)₃) (200 mmol). The solution is heated at reflux for 4-6 h(˜75° C. oil bath temperature). The reaction is monitored by TLC and/orLCMS to completion. After cooling to about 5° C. (ice bath), thereaction is carefully quenched with a 1:1 (v/v) mixture of methanol(MeOH)/water (25 mL) followed by 5 N hydrochloric acid (HCl) (50 mL).The mixture is heated at about 50° C. for about 30-60 min and themajority of the volatile solvents are removed under reduced pressure.Water is added and the aqueous phase is extracted with ethyl acetate(3×). The combined organic extracts are successively washed with asaturated aqueous sodium hydrogencarbonate (NaHCO₃) solution (1×) andwith brine (1×), dried over anhydrous magnesium sulfate (MgSO₄),filtered, and the solvents are evaporated to dryness under reducedpressure. If needed, the crude material is purified by silica gel columnchromatography or is re-crystallized.

Description 7 General Procedure for the Oxidation of Benzylic Alcoholsto Aromatic Aldehydes

Variant A:

Adapting literature known protocols (Parikh, et al., J. Am. Chem. Soc.1967, 89, 5505-5507; and U.S. Pat. No. 8,168,617), to a solution of thealcohol (50 mmol), dimethylsulfoxide (DMSO) (28.5 mL, 400 mmol),triethylamine (Et₃N, TEA) (34.8 mL, 250 mmol) in anhydrousdichloromethane (DCM) (300 mL) is added at 0° C. (ice bath) in smallportions commercial sulfur trioxide-pyridine complex (Pyr.SO₃) (23.9 g,150 mmol). The reaction mixture is stirred with gradual warming to roomtemperature for about 4-12 hours. The reaction is monitored by TLCand/or LCMS to completion. The majority of volatile is evaporated underreduced pressure and the residue is diluted with 2 M hydrochloric acidtill acidic. The aqueous phase is extracted with ethyl acetate (EtOAc)(3×). The combined organic extracts are successively washed with asaturated aqueous sodium hydrogencarbonate (NaHCO₃) solution (1×) andwith brine (1×), dried over anhydrous magnesium sulfate (MgSO₄),filtered, and the solvents are evaporated to dryness under reducedpressure. If needed, the crude material is purified by silica gel columnchromatography or is re-crystallized.

Variant B:

Adapting literature known protocol (Aoyama, et al., Synlett, 1998,35-36), commercial activated manganese(IV) oxide (MnO₂) (250-275 mmol)is added at room temperature to a solution of the benzylic alcohol (25mmol) in dichloromethane (DCM) (100 mL). The reaction mixture is stirredfor 12-24 h. The reaction is monitored by TLC and/or LCMS to completion.The reaction mixture is filtered over a short path of Celite® 545 andthe filtrate is concentrated under reduced pressure. The material isoften of sufficient purity to be used directly in the next step withoutfurther isolation and purification procedures. If needed, the crudematerial is purified by silica gel column chromatography or isre-crystallized.

Variant C:

Adapting a literature known protocol (Corey and Suggs, TetrahedronLett., 1975, 16(31), 2647-2650; and Fujikawa, et al., J. Am. Chem. Soc.,2008, 130, 14533-14543), to a solution of the benzylic alcohol (20 mmol)in dichloromethane (DCM) (100 mL) is added commercial pyridiniumchlorochromate (Pyr⁺CrO₃Cl⁻, PCC) (28-40 mmol). The reaction mixture isheated to reflux (55° C. oil bath temperature) for 1-4 h. The reactionis monitored by TLC and/or LCMS till completion. The reaction is cooledto room temperature. Work-up and product isolation and purification areconducted as described for Variant B.

Description 8 General Procedure for the Addition of MethyltitaniumTrichloride to Aromatic Aldehydes

Adapting a literature known protocol (Reetz, et al., Tetrahedron, 1986,42(11), 2931-2935), titanium tetrachloride (TiCl₄) (10 mmol) is added at−78° C. (dry ice/acetone bath) to cooled anhydrous diethyl ether (Et₂O)(50 mL) under a nitrogen atmosphere resulting in partial precipitationof yellow TiCl₄-bisdiethyl etherate (TiCl₄.2OEt₂). The reaction mixtureis stirred for about 30 min. A solution of methyl lithium (MeLi) (1.6 Min Et₂O) (6.25 mL, 10.0 mmol)) is added slowly which causes a colorchange to dark orange-brown (about 30 min). The reaction mixture isstirred for 30 min and then allowed to warm to about −30° C. A solutionof the aldehyde (10 mmol) in a small amount of Et₂O is added dropwiseand stirred for 1-6 hours at about this temperature (−30° C. to −15°C.). The reaction is monitored by TLC and/or LCMS to completion. Thereaction is quenched by addition of water. The aqueous phase isextracted with additional ethyl acetate (EtOAc) or Et₂O (2×). Thecombined organic extracts are successively washed with a saturatedaqueous sodium hydrogencarbonate (NaHCO₃) solution (1×) and with brine(1×), dried over anhydrous magnesium sulfate (MgSO₄), filtered, and thesolvents are evaporated to dryness under reduced pressure.

Description 9 General Procedure for the Bromination of Benzylic Alcohols

Adapting literature known protocols (Felder, et al., J. Med. Chem.,1970, 13(3), 559-561; and Shen and Jensen, DE Patent No. 2731292A1(1978)), the benzylic alcohol (10 mmol) is dissolved in a solution ofhydrogen bromide in acetic acid (˜1.0 M HBr in HOAc). The brominationsolution is prepared from 33 wt-% HBr (5.71 M in HOAc) (2 mL, 11.4 mmol)and glacial acetic acid (HOAc) (9.5 mL) prior to use. The reactionmixture is heated to about 90-100° C. (oil bath temperature) for 1-12hours under a nitrogen atmosphere. The reaction is monitored by TLCand/or LCMS to completion. The solvents are evaporated to dryness underreduced pressure (rotary evaporator; about 50° C. bath temperature). Theresidue is dissolved in a mixture of ethyl acetate (EtOAc) or diethylether (Et₂O) and hexane. The organic phase is washed with 1 Mhydrochloric acid and the aqueous phase is extracted with the samesolvent mixture (1×). The combined organic extracts are successivelywashed with a saturated aqueous sodium hydrogencarbonate (NaHCO₃)solution (3×) and with brine (1×), dried over anhydrous magnesiumsulfate (MgSO₄), filtered, and the solvents are evaporated to drynessunder reduced pressure. If needed, the crude material is purified bysilica gel column chromatography or is re-crystallized.

Description 10 General Procedure for the Preparation of Nitriles fromBenzylic Bromides

Adapting literature known protocols (Felder, et al., J. Med. Chem.,1970, 13(3), 559-561; and Shen and Jensen, DE Patent No. 2731292A1(1978)), the benzylic bromide (10 mmol) is dissolved inN,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO) or ethanol (EtOH)(10-15 mL). Sodium or potassium cyanide (NaCN or KCN) (11-12 mmol) isadded at once at room temperature. The reaction mixture is heated toabout 75-100° C. (oil bath temperature) for 1 to 12 h under a nitrogenatmosphere. The reaction is monitored by TLC and/or LCMS to completion.EtOH is evaporated to dryness under reduced pressure (rotary evaporator;about 30° C. bath temperature) and the residue is portioned betweenethyl acetate (EtOAc) and a saturated aqueous solution of sodiumhydrogencarbonate (NaHCO₃). If DMF or DMSO is used, the reaction mixtureis directly diluted with EtOAc and a saturated aqueous NaHCO₃ solution.The aqueous phase is extracted with EtOAc (2×). The combined organicextracts are washed with brine (1×), dried over anhydrous magnesiumsulfate (MgSO₄), filtered, and the solvents are evaporated to drynessunder reduced pressure. If needed, the crude material is purified bysilica gel column chromatography or is re-crystallized.

Description 11 General Procedure for the Alkylation of Nitriles

Variant A:

Adapting literature known protocols (Kulig, et al., Pol. J. Chem., 2009,83, 1629-1636; Mann, et al., J. Med. Chem. 1991, 34, 1307-1313; andPeddi, et al., Bioorg. Med. Chem Lett., 2004, 14, 2279-2283), a solutionof the nitrile (10 mmol) in anhydrous tetrahydrofuran (THF) (freshlydistilled over sodium benzophenone ketyl radical) (20-40 mL) is cooledto about −78° C. (dry ice/acetone bath) under a nitrogen atmosphere. Atthis temperature, a commercial solution of lithium diisopropylamide(LDA) (1.8 M in heptane/THF/ethylbenzene) (6.1-6.4 mL, 11-11.5 mmol) isadded dropwise and the reaction mixture is stirred at this temperaturefor about 45-90 min. Depending on the nature of the nitrile substrate,an intense change of color, e.g., dark green-blue, may occur. Commercialalkyl 2-bromoacetate, e.g., BrCH₂CO₂Et, or BrCH₂CO₂tBu) (20 mmol) isadded dropwise in neat form at −78° C. Optionally, the alkylating may bedried over 4 Å molecular sieves (4 Å MS) or may be distilled under anitrogen atmosphere over calcium hydride (CaH₂) prior to use. Thereaction mixture is stirred for about 8-16 hours with gradual warming toroom temperature. The reaction course is followed by TLC and/or LC/MS.The reaction is quenched by addition of 1.0 M hydrochloric acid (HCl).The aqueous phase is extracted with ethyl acetate (EtOAc) (3×). Thecombined organic extracts are successively washed with a saturatedaqueous sodium hydrogencarbonate (NaHCO₃) solution (1×) and brine (1×),dried over anhydrous magnesium sulfate (MgSO₄), filtered, and thesolvents were evaporated to dryness under reduced pressure using arotary evaporator. The crude material is purified by silica gel columnchromatography or is re-crystallized.

Variant B:

Adapting literature known protocols (International ApplicationPublication No. WO 2008/117175; and U.S. Pat. No. 5,015,644), to acooled suspension (about 0° C., ice bath) of sodium hydride (NaH, 60wt-% suspension in mineral oil) (11 mmol, 264 mg (440 mg of the 60 wt-%suspension) in anhydrous dimethylsulfoxide (DMSO) (10-20 mL) under anitrogen atmosphere is dropwise added a solution of the nitrile (10mmol) in anhydrous DMSO) (10-20 mL) and anhydrous diethyl ether (Et₂O)(10-20 mL) (DMSO/Et₂O=2:1 v/v!). The reaction mixture is stirred at thistemperature for about 60-90 min. Depending on the nature of the nitrilesubstrate, an intense change of color, e.g., dark purple, may occur.Commercial alkyl 2-bromoacetate, e.g., BrCH₂CO₂Et, or BrCH₂CO₂tBu) (20mmol) is added dropwise in neat form at 0° C. Optionally, the alkylatingmay be dried over 4A molecular sieves (4A MS) or may be distilled undera nitrogen atmosphere over calcium hydride (CaH₂) prior to use. Thereaction mixture is stirred for about 2-16 h with gradual warming toroom temperature. The reaction course is followed by TLC and/or LC/MS.The reaction is quenched by addition of 1.0 M hydrochloric acid (HCl).Reaction quenching, work-up, product isolation and purification areconducted as described for Variant A.

Description 12 General Procedure for the Reduction of Nitro-Aromates toAnilines

Adapting a literature known protocol (Setamdideh, et al., Orient. J.Chem., 2011, 27(3), 991-996), the nitro-aromatic compound (10 mmol) andfreshly powdered nickel(II) acetate tetrahydrate (Ni(OAc)₂.4H₂O) (2mmol) are dissolved in a mixture of acetonitrile (MeCN) (50 mL) andwater (5 mL) (MeCN/water=1:1 v/v). To aid dissolution of Ni(OAc)₂.4H₂Oin the solvent system, the reaction mixture may be sonicated. Sodiumborohydride (NaBH₄) (40 mmol) is added in small portions (gas evolutionand exotherm!). The reaction mixture is stirred for about 1-5 h at aboutroom temperature. The reaction is monitored by TLC and/or LCMS tocompletion. The reaction mixture is diluted with water and extractedwith ethyl acetate (2-3×). The combined organic extracts are washed withbrine (1×), dried over anhydrous magnesium sulfate (MgSO₄), filtered,and the solvents are evaporated to dryness under reduced pressure. Ifneeded, the crude material is purified by silica gel columnchromatography or is re-crystallized.

Description 13 General Procedure for the Cbz-Protection of Anilines withBenzyl Chloroformate

Adapting literature known protocols (Maciejewski and P. Wipf, ARKIVOC,2011, (vi), 92-119; andYang, et al., Tetrahedron, 2013, 69(15),3331-3337), the aniline (10 mmol) is dissolved in tetrahydrofuran (THF)(20-50 mL). Freshly powdered sodium hydrogencarbonate (NaHCO₃) orpotassium carbonate (K₂CO₃) (11-12 mmol) is added and the reactionmixture is cooled to about 0° C. (ice bath). Neat benzyl chloroformate(CbzCl, BnOCOCl) (11-12 mmol) is added. The reaction mixture is stirredwith warming to room temperature for 1-12 h under a nitrogen atmosphere.The reaction is monitored by TLC and/or LCMS till completion. Thereaction is quenched by addition of water. The majority of the volatiles(THF) are evaporated under reduced pressure (rotary evaporator; ambientbath temperature). The residue is diluted with little 1.0 M hydrochloricacid and the aqueous phase is extracted with ethyl acetate (EtOAc) (3×).The combined organic extracts are successively washed with a saturatedaqueous sodium hydrogencarbonate (NaHCO₃) solution (1×) and with brine(1×), dried over anhydrous magnesium sulfate (MgSO₄), filtered, and thesolvents are evaporated to dryness under reduced pressure. The crudematerial is purified by silica gel column chromatography or isre-crystallized.

Description 14 General Procedure for the Reduction of Aliphatic Nitrilesand In Situ N-Boc Protection

Adapting literature known protocols (Caddick, et al., Tetrahedron, 2003,59(29), 5417-5423), to a solution of the aliphatic nitrile (10 mmol),di-tert-butyl dicarbonate (Boc₂O, Boc-anhydride) (20-30 mmol), andnickel(II) dichloride hexahydrate (NiCl₂.6H₂O) (5 mmol) in methanol(MeOH) (100 mL) at 0° C. (ice bath) is added sodium borohydride (NaBH₄)(80-100 mmol) in small portions. Upon addition of NaBH₄, a blackprecipitate (Ni₂B) is generated immediately and hydrogen gas isgenerated (exotherm!). The reaction mixture is stirred with warming toroom temperature for about 12 hours. The reaction is monitored by TLCand/or LCMS till completion. The reaction is quenched by addition ofwater and 1.0 hydrochloric acid. The aqueous phase is extracted withethyl acetate (EtOAc) (3×). The combined organic extracts aresuccessively washed with a saturated aqueous sodium hydrogencarbonate(NaHCO₃) solution (2×) and with brine (1×), dried over anhydrousmagnesium sulfate (MgSO₄), filtered, and the solvents are evaporated todryness under reduced pressure. The crude material is purified by silicagel column chromatography or is re-crystallized. Optionally, the cruderesidue may be triturated for about 12 h with hexane to remove excessBoc₂O, the solids are collected on a Büchner-funnel and the filtrate isevaporated to dryness under reduced pressure. The evaporated filtratecan be purified by silica gel column chromatography or can bere-crystallized to maximize the overall yield.

Description 15 General Procedure for the Selective N-Boc Protection ofPrimary Amines with Boc₂O

Adapting a literature known protocol (U.S. Pat. No. 8,344,028), to acooled solution of the aniline (10 mmol) in anhydrous dichloromethane(DCM) (20-30 mL) at about 0° C. (ice bath) is added triethylamine (Et₃N,TEA) (12.0 mmol) followed by di-tert-butyl dicarbonate (Boc₂O) (10.5mmol) and a catalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP)(˜0.5 mmol, −5 mol-%). The reaction mixture is stirred with gradualwarming to room temperature for 1-12 h. The reaction is monitored by TLCand/or LCMS to completion. The reaction mixture is diluted with DCM andthe organic phase is washed with a saturated aqueous solution ofammonium chloride (NH₄Cl) (1×) and brine (1×), dried over anhydrousmagnesium sulfate (MgSO₄), filtered, and the solvents are evaporated todryness under reduced pressure. The crude material is purified by silicagel column chromatography or is re-crystallized.

Description 16 General Procedure for the Removal of Cbz-ProtectingGroups by Catalytic Hydrogenolysis

Adapting typical literature known protocols, the Cbz-protected anilineor amine derivative (10 mmol) is dissolved in methanol (MeOH), ethanol(EtOH), ethyl acetate (EtOAc), or mixtures of any of the foregoing(25-50 mL). The heterogeneous catalyst (5 or 10 wt-% palladium oncharcoal containing ˜50 wt-% water) (about 25-50 wt-% with respect tothe Cbz-protected aniline or amine derivative) is added. Optionally, asmall amount of acidic additives, e.g., few drops of HOAc or 1.0 Mhydrochloric acid (HCl) are added to activate the catalyst. Theatmosphere is exchanged to hydrogen (3×evacuation/refill technique) andthe reaction mixture is stirred at room temperature under about 15 psi(H₂-ballon) for 1-12 h. Optionally, the reaction is carried out in astainless steel reactor or a Parr-hydrogenation apparatus if higherpressures of H₂ are required. The reaction is monitored by TLC and/orLCMS till completion. The reaction mixture is filtered over a short plugof Celite® 545, the filtration aid is washed with MeOH, and the combinedfiltrates are evaporated under reduced pressure. The crude material ispurified by silica gel column chromatography or is re-crystallized.

Description 17 General Procedure for the Reductive N-Alkylation

Adapting literature known protocols (Palani, et al., J. Med. Chem.,2005, 48(15), 4746-4749; van Oeveren, Bioorg. Med. Chem. Lett., 2007,17(6), 1527-1531; Delfourne, et al., Bioorg. Med. Chem., 2004, 12(15),3987-3994; Delfourne, et al., J. Med. Chem., 2002, 47(17), 3765-3771;and Jordan, et al., Bioorg. Med. Chem., 2002, 10(8), 2625-2633), to asolution of the aniline (or a suspension of an aniline addition salt,e.g., a hydrochloride salt) (10 mmol) in methanol (MeOH) (30 mL) atabout 5-15° C. (water bath with some ice) is added trifluoroacetic acid(TFA) (15 mL) (Variant A), acetic acid (15-20 mL) (HOAc) (Variant B), or85 wt-% phosphoric acid (H₃PO₄) (Variant C). To the cooled solution, isadded commercial 2-chloroacetaldehyde (ClCH₂CHO) (˜50 wt-% in water,˜7.87 M) (˜6.5 mL, −50 mmol). The reaction mixture is stirred for about15-30 min at this temperature when sodium cyanoborohydride (NaBH₃CN)(2.51 g, 40 mmol) is added in small portions (exothermic hydrogenevolution!). The reaction mixture is stirred for 15-120 min with gradualwarming to room temperature. In some case copious amounts of aprecipitate are generated during the reaction. The reaction course ismonitored by TLC and/or LC/MS till completion. The majority of thevolatiles (Variants A and B) are evaporated under reduced pressure(rotary evaporator; ambient to 35° C. bath temperature). The residue isdissolved in ethyl acetate (EtOAc) and the organic phase is successivelywashed with a saturated aqueous solution of sodium hydrogencarbonate(NaHCO₃) (2×) and brine (x). The organic solution is dried overanhydrous magnesium sulfate (MgSO₄), filtered, and the organic solventswere evaporated to dryness under reduced pressure. If non non-volatileacids are used (Variant C), the reaction mixture is diluted with waterand neutralized (pH 5-7) with solid sodium hydrogencarbonate (NaHCO₃).The aqueous phase is extracted with ethyl acetate (EtOAc) (3×) and thecombined organic extracts are treated as described for Variants A and B.The crude material is purified by silica gel column chromatography or isre-crystallized.

Description 18 General Procedure for the N,N-Bis-(2-Hydroxyethylation)of Anilines with Ethylene Oxide

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Jordan, et al., Bioorg. Med. Chem., 2002, 10(8),2625-2633; Abela Medici, et al, J. Chem. Soc., Perkin Trans. 1, 1997,(20), 2258-2263; Feau, et al., Org. Biomolecular Chem., 2009, 7(24),5259-5270; Springer, et al., J. Med. Chem., 1990, 33(2), 677-681;Taylor, et al., Chem. Biol. Drug Des., 2007, 70(3), 216-226; Buss, etal., J. Fluorine Chem., 1986, 34(1), 83-114; Larden and Cheung,Tetrahedron Lett., 1996, 37(42), 7581-7582; Spreitzer and Puschmann,Monatshefte fiir Chemie, 2007, 138(5), 517-522; Niculesscu-Duvaz, etal., J. Med. Chem., 2004, 47(10), 2651-2658; Weisz, et al., Bioorg. Med.Chem. Lett., 1995, 5(24), 2985-2988; Thorn, et al., J. Org. Chem, 1975,40(11), 1556-1558; Baraldini, et al., J. Med., Chem., 2000, 53(14),2675-2684; Zheng, et al., Bioorg., Med., Chem., 2010, 18(2), 880-886;Gourdi, et al., J., Med., Chem., 1990, 33(4), 1177-1186; Haines, et al.,J. Med. Chem., 1987, 30, 542-547; Matharu, et al., Bioorg. Med. Chem.Lett., 2010, 20, 3688-3691; and Kupczyk-Subotkowska, et al., J. DrugTargeting, 1997, 4(6), 359-370), a mixture of the corresponding aniline(25.0 mmol) in aqueous acetic acid (HOAc) (25-75 vol-%) (25-100 mL) iscooled to about −20° C. (ice/sodium chloride bath) to about 0° C. (icebath). Optionally, the solvent may also glacial acetic acid (HOAc),water, tetrahydrofuran (THF), ethanol (EtOH), 1,4-dioxane (for highertemperature reactions), or mixtures of any of the foregoing. An excessof ethylene oxide (oxirane) (100-400 mmol) is added to the reactionmixture either neat in pre-cooled form or dissolved in any of theforegoing solvents or mixtures thereof. The reaction mixture is stirredat about room temperature for about 12-48 hours. Alternatively, thereaction mixture may be heated in a sealed reaction vessel at 80-140° C.for a similar time. The reaction course is followed by TLC and/or LC/MSand is usually complete when the reaction mixture turns clear. Thesolvents are removed under reduced pressure using a rotary evaporator(40-60° C. water bath temperature). The residue is diluted with ethylacetate (EtOAc), washed with brine, dried over anhydrous magnesiumsulfate (MgSO₄) or sodium sulfate (Na₂SO₄), filtered, and the solventsremoved under reduced pressure using a rotary evaporator to yield thetarget compound, which may be used directly in the next step. The crudematerial may be further purified by silica gel column chromatographyusing EtOAc, methanol (MeOH), dichloromethane and hexanes, or mixturesof any of the foregoing to furnish the purified target compound.Alternatively, the crude target compound may be further purified byre-crystallization.

Description 19 General Procedures for Chlorination ofN,N-Bis(2-Hydroxyethyl)-Groups

Variant A: Chlorination with Thionyl Chloride (SOCl₂)

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Jordan, et al., Bioorg. Med. Chem., 2002, 10(8),2625-2633; Abela Medici, et al., J. Chem. Soc., Perkin Trans. 1, 1997,(20), 2258-2263; Taylor, et al., Chem. Biol. Drug Des., 2007, 70(3),216-226; Dheyongera, Bioorg. Med. Chem. 2005, 13(3), 689-698; Zheng,Bioorg. Med. Chem. 2010, 18(2), 880-886; Gourdi, J. Med. Chem., 1990,33(4), 1177-1186; and Lin, et al., Bioorg. Med. Chem. Lett., 2011,21(3), 940-943), to a solution of thionyl chloride (SOCl₂) (10-75 mmol)in an anhydrous organic solvent, e.g., dichloromethane (DCM), chloroform(CHCl₃), 1,2-dichloroethane (DCE), benzene, or mixtures of any of theforegoing (25-100 mL) is added at a temperature from about 0° C. (icebath) to about room temperature the correspondingN,N-bis(2-hydroxyethyl) derivative (5.0 mmol), either in neat form(portions) or as a solution in a small volume in any of the foregoingsolvents. The reaction mixture is stirred at about room temperature toabout 40° C. or heated to reflux for about 10 minutes to about 3 hours.Optionally, the reaction is carried out using neat SOCl₂ directly as thesolvent. Optionally, the reaction is carried out in the presence of acatalytic amount of zinc chloride (ZnCl₂) (10 mol-% to 40 mol-%) orN,N-dimethylformamide (about 1 to 3 drops) to facilitate the reaction(Squires, et al., J. Org. Chem., 1975, 40(1), 134-136; and Abela Medici,et al, J. Chem. Soc., Perkin Trans. 1, 1997, (20), 2258-2263). Thereaction course is followed by TLC and/or LC/MS till completion.Volatiles (solvents and excess of SOCl₂) are removed under reducedpressure using a rotary evaporator. Optionally, a small amount ofco-solvent, e.g., of benzene, is added to assist in azeotropicco-evaporation and removal of residual excess chlorination agent. Theresidue is diluted with 1.0 M hydrochloric acid (HCl). The aqueous phaseis extracted with ethyl acetate (EtOAc) (3×), and the combined organicextracts are washed with a saturated aqueous solution of sodium hydrogencarbonate (NaHCO₃) (2×) and brine (1×). The organic layer is dried overanhydrous magnesium sulfate (MgSO₄) or sodium sulfate (Na₂SO₄),filtered, and the solvents removed under reduced pressure using a rotaryevaporator. The residue is purified by silica gel column chromatographyusing EtOAc and hexanes mixtures.

Variant B: Chlorination with Phosphoryl Chloride (POCl₃)

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Feau, et al., Org. Biomolecular Chem., 2009, 7(24),5259-5270; Valu, et al., J. Med. Chem., 1990, 33(11), 3014-3019;Baraldini, et al., J. Med., Chem., 2000, 53(14), 2675-2684; Gourdi, etal., J., Med., Chem., 1990, 33(4), 1177-1186; Haines, et al., J. Med.Chem., 1987, 30, 542-547; and Matharu, et al., Bioorg. Med. Chem. Lett.,2010, 20, 3688-3691), to a solution of phosphorus(V) oxychloride(phosphoryl chloride, POCl₃) (10-50 mmol) in an anhydrous organicsolvent, e.g., benzene, acetonitrile, pyridine, or mixtures of any ofthe foregoing (25-100 mL) is added at a temperature from about 0° C.(ice bath) to about room temperature the correspondingN,N-bis(2-hydroxyethyl) derivative (5.0 mmol) either in neat form(portions) or as a solution in a small volume in any of the foregoingsolvents. The remainder of the reaction, work-up, and product isolationare essentially conducted as described in Variant A.

Variant C: Chlorination with Methanesulfonyl Chloride/Pyridine

Adapting literature known protocols (Jordan, et al., Bioorg. Med. Chem.,2002, 10(8), 2625-2633; Abela Medici, et al, J. Chem. Soc., PerkinTrans. 1, 1997, (20), 2258-2263; Springer, et al., J. Med. Chem., 1990,33(2), 677-681; and Larden and Cheung, Tetrahedron Lett., 1996, 37(42),7581-7582), a solution of methanesulfonyl chloride (MsCl) (20.0 mmol) inanhydrous pyridine (about 10 mL) is drop-wise added with stirring and ata temperature of about 0° C. (ice bath) to a solution of thecorresponding N,N-bis(2-hydroxyethyl) derivative (5 mmol) in anhydrouspyridine (about 10 mL). After about 30 minutes, the reaction mixture isheated at 50-100° C. for about 1-3 h. After cooling to room temperature,potential precipitates, if any, e.g., pyridinium methansulfonate, arefiltered off before the solvents are partially removed under reducedpressure using a rotary evaporator. The remainder of the reaction,work-up, and product isolation are essentially conducted as described inVariant A.

Variant D: Chlorination with Triphenylphosphine/Tetrachlorocarbon(PPh₃/CCl₄)

Adapting literature known protocols (Buss, et al., J. Fluorine Chem.,1986, 34(1), 83-114; and Kupczyk-Subotkowska, et al., J. Drug Targeting,1997, 4(6), 359-370), a solution of the correspondingN,N-bis(2-hydroxyethyl) derivative (5 mmol) in anhydrous dichloromethane(DCM) (about 25 mL) containing carbon tetrachloride (CCl₄) (15-25 mmol)is cooled to about 0° C. (ice bath). Alternatively, neat carbontetrachloride (CCl₄) (25 mL) is used as a reaction solvent. The reactionmixture is stirred, and triphenylphosphine (Ph₃P) (10-15 mmol) is addedin portions. The reaction mixture is stirred for about 8-14 h withgradual warming to room temperature. Alternatively, the reaction mixtureis heated at reflux for about 2-6 h. The reaction course is followed byTLC and/or LC/MS till completion. The reaction mixture is cooled to roomtemperature and the solvents are removed under reduced pressure using arotary evaporator. The residue is triturated with diethyl ether (Et₂O)(3×) to remove some of the triphenylphosphine oxide (Ph₃PO). The organicphase is evaporated under reduced pressure using a rotary evaporator.The remainder of the reaction, work-up, and product isolation areconducted as described in Variant A.

Description 20 General Procedure for the Mesylation ofN,N-Bis(2-Hydroxyethyl)-Groups

Variant A:

Adapting literature protocols (Davies, et al., J. Med. Chem. 2005,48(16), 5321-5328; Springer, et al., J. Med. Chem., 1990, 33(2),677-681; Niculesscu-Duvaz, et al., J. Med. Chem., 2004, 47(10),2651-2658; and Yang, et al., Tetrahedron, 2007, 63(25), 5470-5476), to acooled solution (about 0° C. (ice bath)) of the correspondingN,N-bis(2-hydroxyethyl) derivative (5.0 mmol) in anhydrousdichloromethane (DCM) (25-50 mL) are added triethylamine (Et₃N, TEA)(3.48 mL, 2.54 g, 25.0 mmol) or anhydrous pyridine (1.94 mL, 1.98 g,25.0 mmol), and a catalytic amount of 4-N,N-(dimethylamino)pyridine(DMAP) (122 mg, 1.0 mmol, 20 mol-%). Methanesulfonyl anhydride (Ms₂O)(3.48 g, 20.0 mmol) is added portion-wise or as a solution in DCM (5-10mL). The reaction mixture is stirred with gradual warming to roomtemperature for about 8-24 h. The reaction is followed by TLC and/orLC/MS. Solvents are removed under reduced pressure using a rotaryevaporator. The residue is diluted with 1.0 M hydrochloric acid (HCl),and the aqueous phase is extracted with ethyl acetate (EtOAc) (3×). Thecombined organic extracts are washed with a saturated aqueous solutionof sodium hydrogen carbonate (NaHCO₃), and brine, dried over anhydrousmagnesium sulfate (MgSO₄) or sodium sulfate (Na₂SO₄), filtered, and thesolvents are removed under reduced pressure using a rotary evaporator toyield the target compound, which may be used directly in the next step.Alternatively, the crude residue may be further purified by silica gelcolumn chromatography using EtOAc, methanol (MeOH), dichloromethane(DCM), and hexanes, or mixtures of any of the foregoing to furnish thepurified target compound. Alternatively, the crude target compound maybe further purified by re-crystallization.

Variant B:

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Palmer, et al., J. Med. Chem., 1994, 37, 2175-2184;Palmer, et al., J. Med. Chem, 1996, 39(13), 2518-2528; Spreitzer andPuschmann, Monatshefte für Chemie, 2007, 138(5), 517-522; Lin, et al.,Bioorg. Med. Chem. Lett., 2011, 21(3), 940-943; Gourdi, et al., J. Med.Chem., 1990, 33(4), 1177-1186; Ferlin, et al., Bioorg. Med. Chem., 2004,12(4), 771-777; Thorn, et al., J. Org. Chem, 1975, 40(11), 1556-1558;and Coggiola, et al., Bioorg. Med. Chem. Lett., 2005, 15(15),3551-3554), to a cooled solution (about 0° C. (ice bath)) of thecorresponding N,N-bis(2-hydroxyethyl) derivative (5.0 mmol) in anhydrousdichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EtOAc), ora mixture thereof (20-40 mL) are added triethylamine (Et₃N, TEA) (2.1mL, 1.52 g, 15.0 mmol) or anhydrous pyridine (4.04 mL, 3.96 g, 25.0mmol). Methanesulfonyl chloride (MsCl) (0.96 mL, 1.44 g, 12.5 mmol) isadded drop-wise to the reaction mixture. The reaction mixture is stirredfor about 1-2 hours at this temperature. The reaction may be followed byTLC and/or LC/MS. Aqueous work-up and purification by silica gelchromatography are performed as described for Variant A.

Description 21 General Procedure for the Finkelstein Conversion toN,N-Bis(2-Halogenoethyl)-Groups

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Palmer, et al., J. Med. Chem., 1994, 37, 2175-2184;Palmer, et al., J. Med. Chem., 1996, 39(13), 2518-2528; Davies, et al.,J. Med. Chem. 2005, 48(16), 5321-5328; Niculesscu-Duvaz, et al., J. Med.Chem., 2004, 47(10), 2651-2658; Weisz, et al., Bioorg. Med. Chem. Lett.,1995, 5(24), 2985-2988; Thorn, J. Org. Chem, 1975, 40(11), 1556-1558;Lin, et al., Bioorg. Med. Chem. Lett., 2011, 21(3), 940-943; Gourdi, etal., J. Med. Chem. 1990, 33(4), 1177-1186; Yang, et al., Tetrahedron,2007, 63(25), 5470-5476; Ferlin, et al., Bioorg. Med. Chem., 2004,12(4), 771-777; and Coggiola, et al., Bioorg. Med. Chem. Lett., 2005,15(15), 3551-3554), a slurry of the correspondingN,N-bis(2-methylsulfonyloxyethyl) derivative (5.0 mmol) and an alkalimetal halide, e.g., lithium chloride (LiCl), lithium bromide (LiBr),sodium chloride (NaCl), sodium bromide (NaBr), or sodium iodide (NaI)(20-80 mmol) in an anhydrous organic solvent, e.g.,N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), acetone,2-butanone (methyl ethyl ketone, MEK), 3-methyl-2-butanone (isopropylmethyl ketone, MIPK), acetonitrile (MeCN), methanol (MeOH),tetrahydrofuran (THF), ethyl acetate (EtOAc) or a mixture of any of theforegoing (10-30 mL), is stirred at room temperature or heated at50-150° C. for about 1-12 hours. The reaction is followed by TLC and/orLC/MS tocompletion. Solvents are partially or completely removed underreduced pressure using a rotary evaporator. The residue is diluted with1.0 M hydrochloric acid (HCl), and the aqueous phase is extracted withethyl acetate (EtOAc) (3×). The combined organic extracts are washedwith a saturated aqueous solution of sodium hydrogen carbonate (NaHCO₃),and brine, dried over anhydrous magnesium sulfate (MgSO₄) or sodiumsulfate (Na₂SO₄), filtered, and the solvents are removed under reducedpressure using a rotary evaporator to yield the target compound, whichmay be used directly in the next step. Alternatively, the crude residuemay be further purified by silica gel column chromatography using EtOAc,methanol (MeOH), dichloromethane (DCM), and hexanes, or mixtures of anyof the foregoing to furnish the purified target compound. Alternatively,the crude target compound may be further purified by re-crystallization.

Description 22 General Procedure for Deprotection by Acid Hydrolysiswith Strong Aqueous Acids

Adapting literature known protocols (Taylor, et al., Chem. Biol. DrugDes., 2007, 70(3), 216-226; Buss, et al., J. Fluorine Chem., 1986,34(1), 83-114; Abela, et al, J. Chem. Soc., Perkin Trans. 1, 1997, (20),2258-2263; Weisz, et al., Bioorg. Med. Chem. Lett., 1995, 5(24),2985-2988; Zheng, Bioorg., Med., Chem., 2010, 18(2), 880-886; Haines, etal., J. Med. Chem., 1987, 30, 542-547; and Matharu, et al., Bioorg.,Med., Chem., Lett., 2010, 20, 3688-3691), hydrolytic removal ofprotecting groups is conducted through heating a suspension or solutionof the corresponding protected N-mustard (1 mmol) in 2-12 M of anaqueous hydrohalogenic acid (5-10 mL/mmol) or a 20-80 vol-% mixture of a2-12 M of an aqueous hydrohalogenic acid with 1,4-dioxane (5-10 mL/mmol)at an elevated temperature from about 30° C. to about 150° C. (sealedtube) for 1-24 h. The reaction is be followed by TLC and/or LC/MS tocompletion. Organic side products, e.g., phthalic acid or benzoic acid,may be extracted with an organic solvent, e.g., ethyl acetate (EtOAc) orchloroform (CHCl₃). The aqueous solution or organic volatile solventsare evaporated using a rotary evaporator (40° C. to 60° C. water bathtemperature) to yield the crude target product which may be dissolved ina ˜50 vol-% aqueous acetonitrile (MeCN) followed by lyophilization.Where applicable, the crude target compound is further purified byRP-HPLC purification using acetonitrile/water mixtures containing0.05-0.1 vol-% formic acid (FA) or TFA followed by primarylyophilization, optionally in the presence of 1.0 or an excess of anacid capable of forming pharmaceutically acceptable salt additionproducts. Where applicable, the crude material is purified byre-crystallization, titruation, or repeated precipitation.

Description 23 Global Deprotection of Under Anhydrous Conditions withStrong Acids

Variant A:

Adapting literature known protocols (Springer, et al., J. Med. Chem.,1990, 33(2), 677-681; Davies, et al., J. Med. Chem. 2005, 48(16),5321-5328; Niculesscu-Duvaz, et al., J. Med. Chem., 2004, 47(10),2651-2658; Verny and Nicolas, J. Label. Cmpds, Radiopharm., 1988, 25(9),949-955; Thorn, et al., J. Org. Chem, 1975, 40(11), 1556-1558;Baraldini, et al., J. Med. Chem., 2000, 53(14), 2675-2684; Gourdi, etal., J. Med. Chem., 1990, 33(4), 1177-1186; and Kupczyk-Subotkowska, etal., J. Drug Targeting, 1997, 4(6), 359-370), a solution of thecorresponding protected N,N-bis(2-chloroethyl)aryl-substitutedβ-substituted γ-amino acid precursor (1.0 mmol) in neat trifluoroaceticacid (TFA), a mixture of TFA and dichloromethane (DCM) or1,2-dichloroethane (DCE) (90 vol.-% TFA to 90 vol.-% organic solvent),or 98% formic acid (HCO₂H) (10-25 mL/mmol) is stirred at about roomtemperature for about 1-24 h. Optionally, scavengers (2-5 mmol) such astriethysilane (Et₃SiH), triisopropylsilane (iPr₃SiH), thioanisole(PhSMe), or 1,2-dithioethane (HSCH₂CH₂HS) are added to the reactionmixture to suppress unwanted side reactions (Metha, Tetrahedron Lett.,1992, 33(37), 5411-5444). The reaction is be followed by TLC and/oranalytical LC/MS to completion. The solvent is removed under reducedpressure using a rotary evaporator (water bath temperature at about 30°C.). Optionally, residual acid traces are azeotropically removed throughrepeated co-evaporation (5-10×) under reduced pressure using a suitableco-solvent, e.g., ethyl acetate (EtOAc), toluene, or DCM to yield thecrude target compound, which may be used directly in in vitro or in vivoexperiments. Further purification is conducted as described forDescription 22.

Variant B:

Adapting literature known protocols, a solution of the correspondingprotected N,N-bis(2-chloroethyl)aryl-substituted β-substituted γ-aminoacid precursor (1.0 mmol) in 2 M hydrogen chloride in diethyl ether (2.0M HCl in Et₂O) or 4 M hydrogen chloride in 1,4-dioxane (4.0 M HCl in1,4-dioxane) is stirred at about room temperature for about 1-36 h.Optionally scavengers are the same as in Variant A. The reaction is befollowed by TLC and/or analytical LC/MS to completion. The reactionmixture is centrifuged for about 10 min at 3000 rpm, the supernatantdecanted or pipetted off, and the precipitate is suspended in anhydrousEt₂O repeating the centrifugation/washing sequence (2-3×). The crudetarget compound may be used directly in in vitro or in vivo experiments.Further purification is conducted as described for Description 22.

Example 1 4-Amino-3-[3-[bis(2-chloroethyl)amino]phenyl]butanoic acid (1)

Step A: 4-[3-(Bis(2-chloroethyl)amino)phenyl]pyrrolidin-2-one (1a)

Following the General Procedure of Description 17 (Variant A)4-[3-(bis(2-chloroethyl)amino)phenyl]pyrrolidin-2-one (1b) was preparedfrom commercial 4-β-aminophenyl)pyrrolidin-2-one hydrochloride (1.07 g,5.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (3.18 mL,25.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (1.26 g, 20.0 mmol) ina mixture of methanol (MeOH) (15 mL) and trifluoroacetic acid (TFA) (7.5mL). Purification by silica gel column chromatography with ethyl acetate(EtOAc) afforded 1.33 g (88% yield) of the title compound (1a) as acolorless, viscous oil. R_(f): —0.32 (EtOAc). R_(f): —0.41(dichloromethane (DCM)/methanol (MeOH)=95:5 v/v). ¹H NMR (300 MHz,CDCl₃): δ 7.22 (d, J=8.1 Hz, 1H), 6.67 (d, J=7.5 Hz, 1H), 6.59 (dd,J=8.4, 2.4 Hz, 1H), 6.55-6.52 (br. m, 1H), 6.10-6.00 (br. m, 1H),3.82-3.58 (m, 10H), 3.42 (dd, J=9.0, 7.2 Hz, 1H), 2.73 (dd, J=17.1, 9.0Hz, 1H), 2.50 (dd, J=17.1, 9.0 Hz, 1H) ppm. MS (ESI+): m/z=301.05(M+H⁺)⁺.

Step B: 4-Amino-3-[3-[bis(2-chloroethyl)amino]phenyl]butanoic acid (1)

Following the General Procedure of Description 22,4-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]butanoic acid (1) wasprepared from lactam (1a)(4-[3-(bis(2-chloroethyl)amino)phenyl]pyrrolidin-2-one) (878 mg, 2.92mmol) by hydrolysis in concentrated hydrochloric acid (HCl_((aq.)))(about 12 mL) at reflux temperature for about 20 hours to afford 1.05 g(91% yield with respect to the dihydrochloride salt) of the targetcompound (1) (4-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]butanoicacid) as a dihydrochloride salt after isolation using evaporation andlyophilization. Three-hundred-ten (310) mg of the material obtained wasfurther purified by preparative RP-HPLC using a water/acetonitrile/0.1vol-% formic acid gradient to afford 224 mg (72% recovery) of the titlecompound (1) as a dihydrochloride salt with a brownish tint after finallyophilization of the solvents in the presence of an excess of 1.0 Mhydrochloric acid (HCl). The material was of sufficient purity to beused directly and without further isolation and purification proceduresin in vitro and/or in vivo evaluation. LC/UV (from LC/MS): R_(t)=0.84min. 98.1% purity by AUC at λ=254 nm. LC/UV: R_(t)=9.43 min. 95.3%purity by AUC at λ=254 nm. ¹H NMR (300 MHz, D₂O): δ 7.51-7.43 (br. m,1H), 7.35-7.28 (br. m, 3H), 3.90 (t, J=6.0 Hz, 4H), 3.48 (t, J=6.0 Hz,4H), 3.44-3.30 (m, 1H), 3.26 (dd, J=12.9, 5.7 Hz, 1H), 3.14 (dd, J=12.9,10.2 Hz, 1H), 2.78 (dd, J=15.9, 5.4 Hz, 1H), 2.64 (dd, J=16.2, 9.6 Hz,1H) ppm. MS (ESI+): m/z=319.10 (M+H⁺)⁺, (ESI−): m/z=317.00 (M−H⁺)⁻,635.20 (2M−H⁺)⁻. Various batches of mono- or dihydrochloride salts of(1) can be prepared by primary lyophilization of solutions of (1) inaqueous acetonitrile (MeCN) containing either 1.0 eq. of 1.0 Nhydrochloric acid (HCl) or an excess of 1.0 N or higher concentratedhydrochloric acid (HCl).

Example 24-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid (2)

Step A: Methyl (E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (2a)

Following the General Procedure of Description 2 (Variant A), methyl(E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (2a) was prepared fromcommercial 2-methyl-5-nitro-benzaldehyde (3.30 g, 20.0 mmol) (Beech, J.Chem. Soc. (C), 1967, 2374-2375), trimethyl phosphonoacetate (4.04 mL,4.55 g, 25.0 mmol), and anhydrous lithium bromide (LiBr) (2.61 g, 30.0mmol) in a mixture of triethylamine (Et₃N, TEA) (3.83 mL, 2.78 g, 27.5mmol) and acetonitrile (MeCN) (20 mL). Purification by silica gel columnchromatography with a mixture of ethyl acetate (EtOAc) and hexane (1:5v/v) afforded 3.59 g (81% yield) of the title compound (2a) as anoff-white to pale yellow solid. R_(f): —0.30 (EtOAc/hexane=1:4 v/v). ¹HNMR (300 MHz, CDCl₃): δ 8.39 (d, J=2.4 Hz, 1H), 8.11 (dd, J=8.4, 2.4 Hz,1H), 7.92 (d, J=15.9 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H), 6.50 (d, J=15.9Hz, 1H), 3.84 (s, 3H), 2.53 (s, 3H) ppm.

Step B: Methyl 3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate (2b)

Following the General Procedure Description 3, methyl3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate (2b) was prepared frommethyl (E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (2a) (3.38 g, 15.3mmol) in a mixture of nitromethane (MeNO₂) (8.2 mL, 9.34 g, 153 mmol)and acetonitrile (MeCN) (15 mL) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.29 mL, 2.33 g, 15.3 mmol).Purification by silica gel column chromatography with mixtures of ethylacetate (EtOAc) and hexane (1:4 v/v→1:3 v/v) afforded 3.90 g (90% yield)of the title compound (2b) as a pale-yellow oil. R_(f): —0.55(EtOAc/hexane=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.06-8.01 (m, 2H),7.38 (dd, J=9.0, 0.6 Hz, 1H), 4.77 (dd, J=13.2, 6.6 Hz, 1H), 4.65 (dd,J=12.9, 8.1 Hz, 1H), 4.43-4.32 (m, 1H), 3.64 (s, 3H), 2.83-2.81 (br. m,1H), 2.79 (br. d, J=1.2 Hz, 1H), 2.57 (s, 3H) ppm.

Step C: 4-(5-Amino-2-methyl-phenyl)pyrrolidin-2-one (2c)

Following the General Procedure of Description 4,4-(5-amino-2-methyl-phenyl)pyrrolidin-2-one (2c) was prepared frommethyl 3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate (2b) (2.85 g, 10.1mmol), freshly washed active Raney®-3202 nickel (about 10 mL of slurry)in ethanol (EtOH) (85 mL) using a Parr hydrogenation apparatus underabout 50 psi hydrogen pressure. The partially lactamized crude material(¹H NMR analysis (300 MHz, CDCl₃) showed a ratio of non-cyclized form tolactam of about 5:3) was dissolved/suspended in a sealed tube in toluene(about 50 mL) and heated to about 95° C. (oil bath temperature)overnight to complete the lactam formation of non-cyclized material.Purification by silica gel column chromatography with a mixture ofdichloromethane (DCM) and methanol (MeOH) (96:4 v/v) afforded 1.12 g(58% yield) of the title compound (2c) as an off-white solid. R_(f):—0.25 (DCM/MeOH=95:5 v/v). M.p.=146.7° C.-168.7° C. (browning,decomposition). ¹H NMR (300 MHz, CDCl₃): δ 6.95 (d, J=8.4 Hz, 1H), 6.61(d, J=2.4 Hz, 1H), 6.51 (dd, J=8.1, 2.7 Hz, 1H), 6.12-6.00 (br. m, 1H),3.89-3.71 (superimposed, br. m, 2H), 3.60 (br. s, 2H), 3.35 (dd, J=9.0,5.7 Hz, 1H), 2.70 (dd, J=17.8, 9.0 Hz, 1H), 2.43 (dd, J=16.8, 6.9 Hz,1H), 2.22 (s, 3H) ppm. MS (ESI+): m/z=191.10 (M+H⁺)⁺, 381.20 (2M+H⁺)⁺.

Step D: 4-[5-(Bis(2-chloroethyl)amino)-2-methyl-phenyl]pyrrolidin-2-one(2d)

Following the General Procedure of Description 17 (Variant A)4-[5-(bis(2-chloroethyl)amino)-2-methyl-phenyl]pyrrolidin-2-one (2d) wasprepared from 4-(5-amino-2-methyl-phenyl)pyrrolidin-2-one (2c) (1.11 g,5.84 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (3.71 mL,29.2 mmol), and sodium cyanoborohydride (NaBH₃CN) (1.47 g, 23.3 mmol) ina mixture of methanol (MeOH) (20 mL) and trifluoroacetic acid (TFA) (10mL). Purification by silica gel column chromatography with ethyl acetate(EtOAc) afforded 1.72 g (94% yield) of the title compound (2d) as acolorless, viscous oil that solidified to a colorless solid. R_(f):—0.48 (EtOAc). R_(f): —0.43 (dichloromethane (DCM)/methanol (MeOH)=95:5v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.06 (d, J=8.7 Hz, 1H), 6.60 (d, J=2.7Hz, 1H), 6.53 (dd, J=8.4, 2.7 Hz, 1H), 6.22-6.14 (br. m, 1H), 3.93-3.80(m, 1H), 3.79-3.67 (m, 5H), 3.66-3.58 (m, 4H), 3.40 (dd, J=9.3, 6.3 Hz,1H), 3.72 (dd, J=17.1, 9.0 Hz, 1H), 2.45 (dd, J=17.1, 7.8 Hz, 1H), 2.25(s, 3H) ppm. MS (ESI+): m/z=315.10 (M+H⁺)⁺.

Step E: 4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicacid (2)

Following the General Procedure of Description 22,4-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid (2)was prepared from lactam (2d)(4-[5-(bis(2-chloroethyl)amino)-2-methyl-phenyl]pyrrolidin-2-one) (1.01g, 3.20 mmol) by hydrolysis in concentrated hydrochloric acid (HCl)(about 15 mL) at reflux temperature for about 14 hours to afford 1.36 g(˜quantitative yield with respect to the dihydrochloride salt) of thetitle compound (2) as a dihydrochloride salt after isolation usingevaporation and lyophilization. Three-hundred-sixty (360) mg of thematerial thus obtained was purified by preparative RP-HPLC using awater/acetonitrile/0.1 vol-% formic acid gradient to afford 316 mg (88%recovery) of the title compound (2) as an almost colorlessdihydrochloride salt after final lyophilization of the solvents in thepresence of an excess of 1.0 M hydrochloric acid (HCl). LC/UV:R_(t)=10.13 min. ¹H NMR (300 MHz, D₂O): δ 7.37-7.31 (br. m, 2H), 7.26(dd, J=8.4, 2.7 Hz, 1H), 3.90 (t, J=6.0 Hz, 4H), 3.74-3.61 (m, 1H), 3.44(t, J=5.7 Hz, 4H), 3.20 (dd, J=12.9, 6.3 Hz, 1H), 3.10 (dd, J=12.9, 8.4Hz, 1H), 2.81 (dd, J=16.5, 5.1 Hz, 1H), 2.68 (dd, J=16.2, 9.9 Hz, 1H),2.28 (s, 3H) ppm. MS (ESI+): m/z=333.10 (M+H⁺)⁺, (ESI−): m/z=331.00(M−H⁺)⁻. Various batches of mono- or dihydrochloride salts of (2) can beprepared by primary lyophilization of solutions of (2) in aqueousacetonitrile (MeCN) containing either 1.0 eq. of 1.0 N hydrochloric acid(HCl) or an excess of 1.0 N or higher concentrated hydrochloric acid(HCl).

Example 34-Amino-3-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]butanoic acid(3)

Step A: 2,6-Dimethyl-3-nitro-benzaldehyde (3a)

Following the General Procedure of Description 1 (Variant A),2,6-dimethyl-3-nitro-benzaldehyde (3a) was prepared using commercial2,6-dimethylbenzaldehyde (11.5 g, 85.7 mmol) in a mixture of glacialacetic acid (HOAc) (50 mL) and white fuming nitric acid (min. 90 wt-%HNO₃) (100 mL). Aqueous work-up yielded 13.2 g (86% yield) of the targetcompound (3a) as a pale yellow solid. The material obtained was ofsufficient purity to be used directly in the next step without furtherisolation and purification. R_(f): —0.44 (ethyl acetate(EtOAc)/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 10.60 (s, 1H), 7.81(d, J=8.1 Hz, 1H), 7.23 (d, J=8.1 Hz, 1H), 2.65 (s, 3H), 2.62 (s, 3H)ppm.

Step B: Methyl (E)-3-(2,6-dimethyl-3-nitro-phenyl)prop-2-enoate (3b)

Following the General Procedure of Description 2 (Variant A), methyl(E)-3-(2,6-dimethyl-3-nitro-phenyl)prop-2-enoate (3b) was prepared from2,6-dimethyl-3-nitro-benzaldehyde (3a) (13.0 g, 72.6 mmol), trimethylphosphonoacetate (17.6 mL, 19.8 g, 108.8 mmol), and anhydrous lithiumbromide (LiBr) (12.6 g, 145.1 mmol) in a mixture of triethylamine (Et₃N,TEA) (17.7 mL, 12.9 g, 127.0 mmol) and acetonitrile (MeCN) (70 mL).Purification by silica gel column chromatography with a mixture of ethylacetate (EtOAc) and hexane (1:6 v/v) afforded 14.0 g (82% yield) of anyellow and opaque oil consisting of a mixture of about 88.1 wt-% (85mol-% by ¹H NMR) of the target compound (3b) and about 11.9 wt-% (1.9 g,15 mol-% by ¹H NMR) of starting material (3a). The mixture was used inthe next step without further isolation and purification. R_(f): —0.52(EtOAc/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.74 (d, J=16.8 Hz,1H), 7.69 (d, J=8.4 Hz, 1H), 7.18 (d, J=8.7 Hz, 1H), 6.04 (d, J=16.5 Hz,1H), 3.84 (s, 3H), 2.43 (s, 3H), 2.35 (s, 3H) ppm.

Step C: Methyl 3-(2,6-dimethyl-3-nitro-phenyl)-4-nitro-butanoate (3c)

Following the General Procedure of Description 3, methyl3-(2,6-dimethyl-3-nitro-phenyl)-4-nitro-butanoate (3c) was prepared frommethyl (E)-3-(2,6-dimethyl-3-nitro-phenyl)prop-2-enoate (3b) (14.0 g. g,59.5 mmol) in neat nitromethane (MeNO₂) (100 mL, 113.7 g, 1.86 mol) inthe presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (10.0 mL, 10.2g, 66.9 mmol). Purification by silica gel column chromatography withmixtures of ethyl acetate (EtOAc) and hexane (1:4 v/v→1:3 v/v) afforded14.98 g (85% yield) of the title compound (3c) as a yellow oil. Thematerial solidified upon prolonged standing at room temperature to anorange-yellow crystalline material. R_(f): —0.24 (EtOAc/hexane=1:4 v/v).¹H NMR (300 MHz, CDCl₃): δ 7.57 (d, J=8.1 Hz, 1H, major diastereomer),7.49 (d, J=8.4 Hz, 1H, minor diastereomer), 7.19 (d, J=8.1 Hz, 1H, majordiastereomer), 7.11 (d, J=8.1 Hz, 1H, minor diastereomer), 4.95-4.69 (m,3H, both diastereomers), 3.66 (s, 3H, both diastereomers), 2.98-2.78 (m,2H, both diastereomers), 2.57 (s, 3H, major diastereomer), 2.53 (s, 3H,minor diastereomer), 2.48 (s, 3H, minor diastereomer), 2.45 (s, 3H,major diastereomer) ppm. D.r. (by ¹H NMR, 300 MHz, CDCl₃): ˜2.0.

Step D: 4-β-Amino-2,6-dimethyl-phenyl)pyrrolidin-2-one (3d)

Following the General Procedure of Description 4,4-β-amino-2,6-dimethyl-phenyl)pyrrolidin-2-one (3d) was prepared frommethyl 3-(2,6-dimethyl-3-nitro-phenyl)-4-nitro-butanoate (3c) (2.75 g,9.28 mmol), freshly washed active Raney®-3202 nickel (about 9 mL ofslurry) in ethanol (EtOH) (80 mL) using a Parr hydrogenation apparatusunder about 50 psi hydrogen pressure. ¹H NMR analysis (300 MHz, CDCl₃)and TLC analysis showed the presence of both the non-cyclized form andlactam (R_(f) (non-cyclized form): ˜0.14 (dichloromethane (DCM)/methanol(MeOH)=9:1 v/v). The partially lactamized crude material wasdissolved/suspended in a sealed tube in toluene (about 100 mL) andheated to about 95° C. (oil bath temperature) for about 3 h to completethe lactam formation of non-cyclized material (TLC reaction control).After hot filtration, the yellow organic solution was evaporated underreduced pressure using a rotary evaporator to afford an almost colorlesssolid material. The residue was dissolved in DCM and the solvent wasslowly evaporated at room temperature to afford 1.55 g (81% yield) ofthe target compound (3d) as an yellow-brownish crystalline material ofsufficient purity to be used directly in the next step without furtherisolation and purification. R_(f): —0.28 (ethyl acetate (EtOAc)). R_(f):˜0.51 (dichloromethane (DCM)/methanol (MeOH)=9:1 v/v). ¹H NMR (300 MHz,CDCl₃): δ 6.87 (d, J=7.8 Hz, 1H), 6.56 (d, J=7.8 Hz, 1H), 6.20-6.08 (br.m, 1H), 4.25 (ddd, J=17.7, 10.2, 7.8 Hz, 1H), 3.73 (t, J=9.9 Hz, 1H),3.58, 3.48 (m, 3H), 2.69 (dd, J=18.0, 10.8 Hz, 1H), 2.62 (dd, J=18.0,9.6 Hz, 1H), 2.29 (s, 3H), 2.13 (s, 3H) ppm. MS (ESI+): m/z=205.15(M+H⁺)⁺, 409.30 (2M+H⁺)⁺.

Step E:4-[3-(Bis(2-chloroethyl)amino)-2,6-dimethyl-phenyl]pyrrolidin-2-one (3e)

Following the General Procedure for of Description 17 (Variant A)4-[3-(bis(2-chloroethyl)amino)-2,6-dimethyl-phenyl]pyrrolidin-2-one (3e)was prepared from 4-β-amino-2,6-dimethyl-phenyl)pyrrolidin-2-one (3d)(1.55 g, 7.57 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M)(4.81 mL, 37.9 mmol), and sodium cyanoborohydride (NaBH₃CN) (1.97 g,30.3 mmol) in a mixture of methanol (MeOH) (25 mL) and trifluoroaceticacid (TFA) (12.5 mL). Purification by silica gel column chromatographywith ethyl acetate (EtOAc) afforded 1.62 g (65% yield) of the titlecompound (3e) as a colorless viscous oil that solidified to a colorlesssolid. R_(f): ˜0.27 (EtOAc). R_(f): ˜0.61 (dichloromethane(DCM)/methanol (MeOH)=9:1 v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.01 (s, 2H),6.33-6.28 (br. m, 1H), 4.29 (ddd, J=17.4, 10.2, 7.8 Hz, 1H), 3.74 (br.t, J=9.9 Hz, 1H), 3.55 (br. dd, J=9.6, 7.8 Hz, 1H), 3.48-3.42 (m, 4H),3.39-3.32 (m, 4H), 2.76-2.57 (m, 2H), 2.37 (s, 3H), 2.34 (s, 3H) ppm. MS(ESI+): m/z=329.15 (M+H⁺)⁺.

Step F:4-Amino-3-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]butanoic acid(3)

Following the General Procedure of Description 22,4-amino-3-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]butanoic acid(3) was prepared from4-[3-(bis(2-chloroethyl)amino)-2,6-dimethyl-phenyl]pyrrolidin-2-one (3e)(765 mg, 2.32 mmol) by hydrolysis in concentrated hydrochloric acid(HCl) (about 10 mL) at reflux temperature for about 32 hours to afford856 mg (88% yield with respect to the dihydrochloride salt) of the titlecompound (3) as a dihydrochloride salt after isolation using evaporationand lyophilization. Four-hundred-fifty-nine (459) mg of the materialthus obtained was purified by preparative RP-HPLC using awater/acetonitrile/0.1 vol-% formic acid gradient to afford 173 mg (38%recovery) of the title compound (3) as an off-white dihydrochloride saltafter final lyophilization of the solvents in the presence of an excessof 1.0 M hydrochloric acid (HCl). LC/UV: R_(t)=8.00 min. 95.7% purity byAUC at λ=254 nm. ¹H NMR (300 MHz, CD₃OD): δ 7.46-7.36 (br. m, 1H, bothdiastereomers), 7.33-7.20 (br. m, 1H, both diastereomers), 4.17-4.00(br. m, 1H, both diastereomers), 3.90-3.75 (br. m, 4H, bothdiastereomers), 3.12-2.97 (br. m, 5H, both diastereomers), 2.90-2.75(br. m, 1H, both diastereomers, superimposed with CD₃OH-signal),3.12-2.96 (br. m, 1H, both diastereomers), 2.90-2.75 (br. m, 1H, bothdiastereomers), 2.61, 2.55, 2.51, 2.47 (4s, 6H, both diastereomers) ppm.D.r. (by ¹H NMR, 300 MHz, CD₃OD): ˜1.0. MS (ESI+): m/z=347.15 (M+H⁺)⁺,695.35 (2M+H⁺)⁺. (ESI−): m/z=345.05 (M−H⁺)⁻, 693.30 (2M−H⁺)⁻. Variousbatches of mono- or dihydrochloride salts of (3) were prepared byprimary lyophilization of solutions of (3) in aqueous acetonitrile(MeCN) containing either 1.0 eq. of 1.0 N hydrochloric acid (HCl) or anexcess of 1.0 N or higher concentrated hydrochloric acid (HCl).

Example 44-Amino-3-[3-[bis(2-chloroethyl)amino]phenyl]-3-methyl-butanoic acid (4)

Step A: Methyl (E/Z)-3-β-nitrophenyl)but-2-enoate (4a)

Following the General Procedure of Description 2 (Variant B), methyl(E/Z)-3-β-nitrophenyl)but-2-enoate (4a) was prepared from commercial3′-nitroacetophenone (m-nitroacetophenone) (3.44 g, 20.44 mmol),trimethyl phosphonoacetate (3.71 mL, 4.17 g, 22.90 mmol), and sodiumhydride (NaH) (550 mg, 22.91 mmol, 916 mg of 60 wt-% suspension inmineral oil) in anhydrous N,N-dimethylformamide (DMF) (70 mL). Aqueouswork-up yielded 5.23 g of the target compound (4a) as yellow needles.The material obtained consisted of a mixture of geometric isomers((E)/(Z)-isomeric mixture) of methyl (E)-3-β-nitrophenyl)but-2-enoate(major isomer) and methyl (Z)-3-β-nitrophenyl)but-2-enoate(minor isomer)in a ratio of approximately 4:1 by ¹H NMR (300 MHz, CDCl₃) and smallamounts of residual solvents and trimethyl phosphonoacetate(HWE-reagent). The mixture was of sufficient purity to be used directlyin the next step without further isolation and purification. R_(f)((E)-Isomer): ˜0.43 (EtOAc/hexane=1:4 v/v); R_(f) ((Z)-Isomer): ˜0.32(EtOAc/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃, both isomers): δ 8.32(br. dd, J=2.1, 0.9 Hz, 1H, major isomer), 8.22 (ddd, J=8.4, 2.1, 1.2Hz, 1H, major isomer, partially superimposed with minor isomer),8.08-7.99 (br. m, 1H, minor isomer), 7.79 (ddd, J=8.1, 2.1, 1.2 Hz, 1H,major isomer, partially superimposed with minor isomer), 7.56 (t, J=8.1Hz, 1H, major isomer partially superimposed with minor isomer), 6.20 (q,J=1.2 Hz, 1H, major isomer), 6.01 (q, J=1.2 Hz, 1H, minor isomer), 3.78(s, 3H, major isomer), 3.57 (s, 3H, minor isomer), 2.61 (d, J=1.2 Hz,3H, major isomer), 2.21 (d, J=1.5 Hz, 3H, minor isomer) ppm. D.r. (¹HNMR, 300 MHz, CDCl₃): ˜4.

Step B: Methyl 3-methyl-4-nitro-3-β-nitrophenyl)butanoate (4b)

Following the General Procedure of Description 3, methyl3-methyl-4-nitro-3-β-nitrophenyl)butanoate (4b) was prepared from methyl(E/Z)-3-β-nitrophenyl)but-2-enoate (4a) (˜4.78 g. g, ˜18.44 mmol) inneat nitromethane (MeNO₂) (100 mL, 113.7 g, 1.86 mol) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (10.0 mL, 10.2 g, 66.9 mmol).Purification by silica gel column chromatography with mixtures of ethylacetate (EtOAc) and hexane (1:4 v/v→1:3 v/v) afforded 353 mg (9% yield)of recovered starting material (4a) (pure methyl(E)-3-β-nitrophenyl)but-2-enoate) and 3.21 g (68% yield based onrecovered starting material) of the title compound (4b) an orangeviscous oil. R_(f): ˜0.43 (EtOAc/hexane=1:2 v/v). ¹H NMR (300 MHz,CDCl₃): δ 8.23 (br. t, J=2.1 H, 1H), 8.17 (ddd, J=8.1, 2.4, 1.2 Hz, 1H),7.68 (ddd, J=7.8, 1.8, 0.9 Hz, 1H), 7.56 (t, J=7.8 Hz, 1H), 4.97 (d,J=11.7 Hz, 1H), 4.92 (d, J=11.7 Hz, 1H), 3.63 (s, 3H), 3.00 (d, J=15.9Hz, 1H), 2.96 (d, J=16.2 Hz, 1H), 1.70 (s, 3H) ppm.

Step C: 4-β-Aminophenyl)-4-methyl-pyrrolidin-2-one (4c)

Following the General Procedure of Description 4,4-β-aminophenyl)-4-methyl-pyrrolidin-2-one (4c) was prepared from methyl3-methyl-4-nitro-3-β-nitrophenyl)butanoate (4b) (1.41 g, 5.00 mmol),freshly washed active Raney®-3202 nickel (about 5 mL of slurry) inethanol (EtOH) (70 mL) using a Parr hydrogenation apparatus under about50 psi hydrogen pressure to afford 880 mg (93% yield) of the targetcompound (4c) as an off-white to beige powder. ¹H NMR analysis (300 MHz,CD₃OD) and TLC analysis showed that the material thus obtained consistedexclusively of the lactam (cyclized form). The material was ofsufficient purity to be used directly and without further isolation andpurification in the next step. M.p.: R_(f): ˜0.51 (dichloromethane(DCM)/methanol (MeOH)=9:1 v/v). ¹H NMR (300 MHz, CD₃OD): δ7.06 (br. t,J=8.1 Hz, 1H), 6.64-6.54 (m, 3H), 3.60 (d, J=9.9 Hz, 1H), 3.46 (d, J=9.9Hz, 1H), 2.73 (d, J=16.5 Hz, 1H), 2.37 (d, J=16.5 Hz, 1H), 1.42 (s, 3H)ppm. MS (ESI+): m/z=191.10 (M+H⁺)⁺, 381.20 (2M+H⁺)⁺.

In a second reaction of the same scale, under comparable conditions, andusing comparable procedures, the initially isolated material consistedexclusively of the non-cyclized form (methyl4-amino-3-β-aminophenyl)-3-methyl-butanoate) MS (ESI+): m/z=223.20(M+H)⁺. The crude material was dissolved/suspended in a sealed tube intoluene (about 25 mL) and heated to about 100° C. (oil bath temperature)for overnight to facilitate lactam formation (TLC reaction control). Theorganic solution was evaporated under reduced pressure using a rotaryevaporator to afford a beige-brown waxy solid that was further purifiedby silica gel column chromatography using mixtures of dichloromethane(DCM) and methanol (MeOH) as eluent (DCM/MeOH=95:5-93:7) to afford thetitle compound (4c) as a beige solid. The analytical data correspondedto the proposed structure and was identical to that obtained from thedirect lactamization method.

Step D: 4-[3-(Bis(2-chloroethyl)amino)phenyl]-4-methyl-pyrrolidin-2-one(4d)

Following the General Procedure of Description 17(Variant A)4-[3-(bis(2-chloroethyl)amino)phenyl]-4-methyl-pyrrolidin-2-one (4d) wasprepared from 4-β-aminophenyl)-4-methyl-pyrrolidin-2-one (4c) (571 mg,3.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (1.91 mL,15.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (754 mg, 12.0 mmol) ina mixture of methanol (MeOH) (10 mL) and trifluoroacetic acid (TFA) (5.0mL). Purification by silica gel column chromatography with ethyl acetate(EtOAc) afforded 876 mg (92% yield) of the title compound (4d) as acolorless oil that solidified to a colorless solid. R_(f): ˜0.38(EtOAc). R_(f): ˜0.58 (dichloromethane (DCM)/methanol (MeOH)=9:1 v/v).¹H NMR (300 MHz, CDCl₃): δ 7.22 (d, J=8.1 Hz, 1H), 6.64-6.55 (m, 2H),6.47 (t, J=1.8 Hz, 1H), 6.28-621 (br. m, 1H), 3.77-3.59 (m, superimposedsignals, 9H), 3.48 (dd, J=9.3, 1.2 Hz, 1H), 2.76 (d, J=16.2 Hz, 1H),2.44 (d, J=16.5 Hz, 1H), 1.49 (s, 3H) ppm. MS (ESI+): m/z=329.15(M+H⁺)⁺.

Step E: 4-Amino-3-[3-[bis(2-chloroethyl)amino]phenyl]-3-methyl-butanoicacid (4)

Following the General Procedure of Description 22,4-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]-3-methyl-butanoic acid (4)was prepared from4-[3-(bis(2-chloroethyl)amino)phenyl]-4-methyl-pyrrolidin-2-one (4d)(671 mg, 2.13 mmol) by hydrolysis in concentrated hydrochloric acid(HCl) (about 12 mL) at reflux temperature for about 15 hours to affordthe title compound (4) as a dihydrochloride salt after isolation usingevaporation and lyophilization. Two-hundred-fifty (250) mg of thematerial thus obtained was purified by preparative RP-HPLC using awater/acetonitrile/0.1 vol-% formic acid gradient to afford 190 mg (76%recovery) of the title compound (4) as an almost colorlessdihydrochloride salt after final lyophilization of the solvents in thepresence of an excess of 1.0 M hydrochloric acid (HCl). The material wasof sufficient purity to be used directly and without further isolationand purification procedures in in vitro and/or in vivo evaluation. LC/UV(from LC/MS): R_(t)=0.98 min. 98.1% purity by AUC at λ=254 nm. ¹H NMR(300 MHz, CD₃OD): δ 7.52 (t, J=8.1 Hz, 1H), 7.47-7.41 (br. m, 1H),7.37-7.24 (br. M, 2H), 4.03 (br. t, J=6.6 Hz, 4H), 3.68 (br. t, J=6.3Hz, 4H), 3.48 (d, J=13.2 Hz, 1H), 3.42 (d, J=13.2 Hz, 1H), 2.91 (d,J=15.3 Hz, 1H), 2.84 (d, J=15.3 Hz, 1H), 1.62 (s, 3H) ppm. MS (ESI+):m/z=333.10 (M+H⁺)⁺. (ESI−): m/z=331.10 (M−H⁺)⁻, 665.35 (2M−H⁺)⁻. Variousbatches of mono- or dihydrochloride salts of (4) were prepared byprimary lyophilization of solutions of (4) in aqueous acetonitrile(MeCN) containing either 1.0 eq. of 1.0 N hydrochloric acid (HCl) or anexcess of 1.0 N or higher concentrated hydrochloric acid (HCl).

Example 54-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (5) Method A

Step A: 1-(2-Methyl-5-nitro-phenyl)ethanone (5a)

Following the General Procedure of Description 1 (Variant A),1-(2-methyl-5-nitro-phenyl)ethanone (5a) was prepared commercial1-(o-tolyl)ethanone (2′-methylacetophenone) (12.3 g, 91.9 mmol) in amixture of glacial acetic acid (HOAc) (40 mL) and white fuming nitricacid (min. 90 wt-% HNO₃) (80 mL). After aqueous work-up, the crudematerial consisted mainly of a mixture of two regioisomers1-(2-methyl-5-nitro-phenyl)ethanone (5a) and1-(2-methyl-3-nitro-phenyl)ethanone (5a′), which were separated bysilica gel column chromatography using a mixture of ethyl acetate(EtOAc) and hexane (EtOAc/hexane=1:9) as an eluent to afford 5.30 g (32%yield) the title compound (5a) and the corresponding regioisomer (5a′)(1-(2-methyl-3-nitro-phenyl)ethanone) as a yellow solid (see alsoExample 6). R_(f)(5a): ˜0.51 (ethyl acetate (EtOAc)/hexane=1:4 v/v).R_(f)(5a): ˜0.25 (ethyl acetate (EtOAc)/hexane=1:9 v/v). ¹H NMR (300MHz, CDCl₃, (5a)): δ 8.55 (d, J=2.4 Hz, 1H), 8.22 (dd, J=8.7, 2.7 Hz,1H), 7.44 (d, J=8.7 Hz, 1H), 2.67 (s, 3H), 2.63 (s, 3H) ppm. R_(f)(5a′):˜0.34 (ethyl acetate (EtOAc)/hexane=1:4 v/v). R_(f)(5a′): ˜0.16 (ethylacetate (EtOAc)/hexane=1:9 v/v). ¹H NMR (300 MHz, CDCl₃, (5a′)): δ 7.84(dd, J=7.8, 1.2 H, 1H), 7.72 (dd, J=7.5, 1.2 Hz, 1H), 7.46-7.38 (m, 1H),2.61 (s, 3H), 2.52 (s, 3H) ppm.

Step B: 1-(2-Methyl-5-nitro-phenyl)ethanol (5b)

Following the General Procedure of Description 5,1-(2-methyl-5-nitro-phenyl)ethanol (5b) was prepared from1-(2-methyl-5-nitro-phenyl)ethanone (5a) (5.30 g, 29.6 mmol) and sodiumborohydride (NaBH₄) (567 mg, 15.0 mmol) in ethanol (EtOH) (50 mL). Afteraqueous work-up, 5.51 g (˜quantitative yield) of the title compound (5b)were obtained as a yellow viscous oil that solidified upon standing atroom temperature. The material was of sufficient purity to be useddirectly and without additional isolation and purification in the nextstep. R_(f): ˜0.22 (ethyl acetate (EtOAc)/hexane=1:4 v/v). ¹H NMR (300MHz, CDCl₃): δ 8.41 (d, J=2.4 Hz, 1H), 8.00 (dd, J=8.7, 2.7 Hz, 1H),7.27 (d, J=8.7 Hz, 1H), 5.16 (q, J=6.3 Hz, 1H), 2.43 (s, 3H), 1.49 (d,J=6.3 Hz, 3H) ppm.

Step C: 2-(1-Bromoethyl)-1-methyl-4-nitro-benzene (5c)

Following the General Procedure of Description 9,2-(1-bromoethyl)-1-methyl-4-nitro-benzene (5c) was prepared from1-(2-methyl-5-nitro-phenyl)ethanol (5b) (5.51 g, 29.6 mmol) and 1.0 Mhydrogen bromide in acetic acid (HBr/HOAc) (prepared from 33 wt-% HBr inHOAc (5.70 mL, 32.6 mmol) and glacial acetic acid (26.9 mL)). Afteraqueous work-up, 6.95 g (96% yield) of the title compound (5c) wereobtained as a brown-colored viscous oil that crystallized to a beigesolid upon standing at room temperature. The material was of sufficientpurity to be used directly and without additional isolation andpurification in the next step. R_(f): ˜0.71 (ethyl acetate(EtOAc)/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.40 (d, J=2.1 Hz,1H), 8.05 (dd, J=8.1, 2.1 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H), 5.35 (q,J=7.2 Hz, 1H), 2.51 (s, 3H), 2.13 (d, J=6.9 Hz, 3H) ppm.

Step D: 2-(2-Methyl-5-nitro-phenyl)propanenitrile (5d)

Following the General Procedure of Description 10,2-(2-methyl-5-nitro-phenyl)propanenitrile (5d) was prepared from2-(1-bromoethyl)-1-methyl-4-nitro-benzene (5c) (6.95 g, 28.5 mmol) andsodium cyanide (NaCN) (1.66 g, 33.7 mmol) in anhydrousN,N-dimethylformamide (DMF) (90 mL). Purification by silica gel columnchromatography with ethyl acetate (EtOAc)/hexane mixtures(EtOAc/hexane=2:5-1:3 v/v) afforded 3.93 g (74% yield) of the titlecompound (5d) as a reddish-brownish solid. R_(f): ˜0.38 (ethyl acetate(EtOAc)/hexane=1:4 v/v). R_(f): ˜0.58 (ethyl acetate (EtOAc)/hexane=1:2v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.32 (d, J=2.1 Hz, 1H), 8.11 (dd,J=8.1, 2.1 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 4.10 (q, J=7.2 Hz, 1H), 2.50(s, 3H), 1.70 (d, J=7.2 Hz, 3H) ppm.

Step E: Ethyl 3-cyano-3-(2-methyl-5-nitro-phenyl)butanoate (5e)

Following the General Procedure of Description 11 (Variant A), ethyl3-cyano-3-(2-methyl-5-nitro-phenyl)butanoate (5e) was prepared from2-(2-methyl-5-nitro-phenyl)propanenitrile (5d) (641 mg, 3.37 mmol) bydeprotonation with a commercial solution of lithium diisopropylamide(LDA) (1.8 M in heptane/THF/ethylbenzene) (2.06 mL, 3.71 mmol) andsubsequent alkylation with commercial, neat ethyl 2-bromoacetate(BrCH₂CO₂Et) (746 μL, 1.13 g, 6.74 mmol; dried over 4A molecular sieves(4A MS) prior to use) in anhydrous tetrahydrofuran (THF) (freshlydistilled over sodium benzophenone ketyl radical) (17 mL). Purificationby silica gel column chromatography using an ethyl acetate(EtOAc)/hexane mixture (EtOAc/hexane=1:4 v/v) afforded 735 mg of anorange oil. ¹H NMR analysis (300 MHz, CDCl₃) of the purified productshowed an inseparable mixture in a molar ratio of the title compound(5e) to starting material (5d) of about 63/37, corresponding toapproximately 463 mg (approximately 50% yield) of the title compound(5e). R_(f): ˜0.54 (ethyl acetate (EtOAc)/hexane=1:2 v/v)¹H NMR (300MHz, CDCl₃): δ 8.19 (d, J=2.1 Hz, 1H), 8.11 (dd, J=8.4, 2.4 Hz, 1H),7.42 (d, J=8.4 Hz, 1H), 4.10 (q, J=7.2 Hz, 2H), 3.14 (d, J=15.6 Hz, 1H),3.01 (d, J=15.9 Hz, 1H), 2.78 (s, 3H), 2.05 (s, 3H), 1.18 (t, J=7.2 Hz,3H) ppm.

Step F: 4-β-Amino-6-methyl-phenyl)-4-methyl-pyrrolidin-2-one (5f)

Following the General Procedure for of Description 4,4-β-amino-6-methyl-phenyl)-4-methyl-pyrrolidin-2-one (5f) was preparedfrom a mixture of ethyl 3-cyano-3-(2-methyl-5-nitro-phenyl)butanoate(5e) and 2-(2-methyl-5-nitro-phenyl)propanenitrile (5d) (containing ˜463mg, ˜1.68 mmol of (5e)), freshly washed active Raney®-3202 nickel (about6 mL of slurry) in ethanol (EtOH) (60 mL) using a Parr hydrogenationapparatus under about 60 psi hydrogen pressure. ¹H NMR analysis (300MHz, CDCl₃) and TLC analysis showed that the reaction mixture consistedmainly of partially reduced starting material ethyl3-(5-amino-2-methyl-phenyl)-3-cyano-butanoate (5f″), completely reducednon-cyclized starting material ethyl4-amino-3-(5-amino-2-methyl-phenyl)-3-methyl-butanoate (5f′), and thetitle compound 4-β-amino-6-methyl-phenyl)-4-methyl-pyrrolidin-2-one(5f). A concentrated filtration alcoholic solution (about 10 mL) of thecrude reaction mixture was stirred for about 2 hours at about 60-80° C.to facilitate lactamization of non-cyclized reduction product (5f′) tothe title compound (5f) together with slow evaporation of residualsolvent. The material thus obtained was further purified by silica gelcolumn chromatography using dichloromethane (DCM) and methanol (MeOH)mixtures (DCM/MeOH=96:4-95:5 v/v) as eluents to afford 250 mg (71%yield) of the target compound (5f) as a colorless solid. R_(f): ˜0.60(DCM/MeOH=9:1 v/v). R_(f): ˜0.38 (DCM/MeOH=95:5 v/v). ¹H NMR (300 MHz,CDCl₃): δ 6.93 (d, J=8.1 Hz, 1H), 6.51 (dd, J=8.1, 2.4 Hz, 1H), 6.40 (d,J=2.1 Hz, 1H), 6.10-6.00 (br. m, 1H), 3.75 (br. d, J=9.3 Hz, 1H), 3.59(br. dd, J=9.0, 1.8 Hz, 1H, superimposed), 3.59 (br. s, 2H,superimposed), 2.82 (d, J=16.2 Hz, 1H), 2.54 (d, J=16.2 Hz, 1H), 2.25(s, 3H), 1.46 (s, 3H) ppm. MS (ESI+): m/z=205.20 (M+H⁺)⁺, 409.3(2M+H⁺)⁺.

Step G:4-[5-(Bis(2-chloroethyl)amino)-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(5g)

Following the General Procedure of Description 17 (Variant A),4-[5-(bis(2-chloroethyl)amino)-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(5g) was prepared from4-β-amino-6-methyl-phenyl)-4-methyl-pyrrolidin-2-one (5f) (250 mg, 1.22mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (777 μL, 6.11mmol), and sodium cyanoborohydride (NaBH₃CN) (307 mg, 4.89 mmol) in amixture of methanol (MeOH) (4.0 mL) and trifluoroacetic acid (TFA) (2.0mL). Purification by silica gel column chromatography with ethylacetate(EtOAc) afforded 403 mg (˜quantitative yield) of the title compound (5g)as a colorless oil that solidified to a colorless solid. R_(f): ˜0.42(EtOAc). R_(f): ˜0.54 (dichloromethane (DCM)/methanol (MeOH)=9:1 v/v).¹H NMR (300 MHz, CDCl₃): δ 7.02 (d, J=8.4 Hz, 1H), 6.51 (dd, J=8.4, 2.7Hz, 1H), 6.42-6.35 (br. m, 2H), 3.77 (br. d, J=9.3 Hz, 1H), 3.73-3.57(br. m, 9H, superimposed), 2.85 (d, J=15.9 Hz, 1H), 2.58 (d, J=16.2 Hz,1H), 2.26 (s, 3H), 1.48 (s, 3H) ppm. MS (ESI+): m/z=329.15 (M+H⁺)⁺.

Step H:4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (5)

Following the General Procedure of Description 22,4-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (5) was prepared from4-[5-(bis(2-chloroethyl)amino)-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(5g) (401 mg, 1.22 mmol) by hydrolysis in concentrated hydrochloric acid(HCl) (about 15 mL) at reflux temperature for about 24 h to afford 494mg (96% yield) the title compound (5) as a dihydrochloride salt afterisolation using evaporation and lyophilization together with about 50%of starting material (5g). Two-hundred-fifty (250) mg of the materialthus obtained was purified by preparative RP-HPLC using awater/acetonitrile/0.1 vol-% formic acid gradient to afford 65 mg (52%recovery considering purity of the crude reaction product) of the titlecompound (5) as a slightly brown dihydrochloride salt after finallyophilization of the solvents in the presence of an excess of 1.0 Mhydrochloric acid (HCl). The material was of sufficient purity to beused directly and without further isolation and purification proceduresin in vitro and/or in vivo evaluation. LC/UV (from LC/MS): R_(t)=1.20min. 99.0% purity by AUC at λ=254 nm. ¹H NMR (300 MHz, CD₃OD): δ7.48-7.31 (br. m, 3H), 4.04 (br. t, J=6.6 Hz, 4H), 3.70-3.59 (br. m,5H), 3.41 (d, J=13.5 Hz, 1H), 3.10 (d, J=16.2 Hz, 1H), 2.87 (d, J=15.9Hz, 1H), 2.60 (s, 3H), 1.77 (s, 3H) ppm. MS (ESI+): m/z=347.15 (M+H⁺)⁺.(ESI−): m/z=345.05 (M−H⁺)⁻, 693.30 (2M−H⁺)⁻. Various batches of mono- ordihydrochloride salts of (5) were prepared by primary lyophilization ofsolutions of (5) in aqueous acetonitrile (MeCN) containing either 1.0eq. of 1.0 N hydrochloric acid (HCl) or an excess of 1.0 N or higherconcentrated hydrochloric acid (HCl).

Method B

Step I: (2-Methyl-5-nitro-phenyl)methanol (5i)

Following the General Procedure of Description 6,2-methyl-5-nitro-phenyl)methanol (5i) was prepared from commercial2-methyl-5-nitro benzoic acid (50.0 g, 276 mmol) with boranedimethylsulfide complex (2.0 M BH₃. SMe₂ in THF) (166 mL, 332 mmol) inanhydrous tetrahydrofuran (400 mL) to yield 44.0 g (˜quantitative yield)of the target compound (5i) as a pale yellow solid which was ofsufficient purity to be used directly in the next step without furtherisolation and purification procedures. R_(f): ˜0.50 (EtOAc/Hxn=1:1 v/v).¹H NMR (300 MHz, CDCl₃): δ 8.30 (d, J=2.4 Hz, 1H), 8.05 (dd, J=8.4, 2.4Hz, 1H), 7.31 (d, J=8.1 Hz, 1H), 4.78 (d, J=5.1 Hz, 2H), 2.41 (s, 3H),1.87 (br. t, J=5.1 Hz, 1H) ppm.

Step J: 2-Methyl-5-nitro-benzaldehyde (5j)

Following the General Procedure of Description 7 (Variant A),2-methyl-5-nitro-benzaldehyde (5j) (Beech, J. Chem. Soc. (C), 1967,2374-2375) was prepared from 2-methyl-5-nitro-phenyl)methanol (5i) (16.3g, 97.3 mmol) in the presence of dimethylsulfoxide (DMSO) (56.8 mL, 62.6g, 0.80 mol), triethylamine (TEA, Et₃N) (69.5 mL, 50.6 g, 0.50 mmol),and sulfur trioxide pyridine complex (SO₃.pyridine) (47.8 g, 0.30 mol)in dichloromethane (600 mL). Purification by silica gel columnchromatography using a mixture of ethyl acetate (EtOAc) and hexane(EtOAc/hexane=1:4 v/v) afforded 12.6 g (78% yield) of the targetcompound (5j) as a yellow-beige solid.

Following the General Procedure of Description 7 (Variant B),2-methyl-5-nitro-benzaldehyde (5j) (Beech, J. Chem. Soc. (C), 1967,2374-2375) was prepared from 2-methyl-5-nitro-phenyl)methanol (5i) (4.03g, 24.1 mmol) in the presence of manganese dioxide (MnO₂) (22 g, 254mmol) in dichloromethane (DCM) (100 mL). Work-up afforded 3.56 g (89%yield) of the target compound (5j) as a pale yellow to beige solid. Thematerial was of sufficient purity to be used directly in the next stepwithout further isolation and purification procedures.

Following the General Procedure of Description 7 (Variant C),2-methyl-5-nitro-benzaldehyde (5j) (Beech, J. Chem. Soc. (C), 1967,2374-2375) was prepared from 2-methyl-5-nitro-phenyl)methanol (5i) (5.00g, 29.9 mmol) in the presence of pyridinium chlorochromate (PCC) (9.02g, 41.9 mmol) in dichloromethane (DCM) (150 mL). Purification by silicagel column chromatography using mixtures of ethyl acetate (EtOAc) andhexane (EtOAc/hexane=1:4 v/v→EtOAc/hexane=1:4 v/v) afforded 4.67 g (94%yield) of the target compound (5j) as a yellow-beige solid. R_(f): ˜0.76(EtOAc/Hxn=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 10.32 (s, 1H), 8.65 (dd,J=2.7 Hz, 1H), 8.31 (dd, J=8.4, 2.4 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H),2.79 (s, 3H) ppm.

Step K: 1-(2-Methyl-5-nitro-phenyl)ethanol (5k)

Following the General Procedure of Description 8, MeTiCl₃ was freshlyprepared prior to use from commercial titanium tetrachloride (TiCl₄)(10.0 mL, 17.3 g, 91.0 mmol) and methyl lithium (MeLi) (1.6 M in Et₂O,57.0 mL, 91.0 mmol) in anhydrous diethyl ether (Et₂O) (400 mL). Asolution of 2-methyl-4-nitro-benzaldehyde (5j) (12.4 g, 75.01 mmol) inanhydrous Et₂O (200 mL) was added. Aqueous work-up afforded 13.7 g (99%yield) of the target compound (5k) as a yellow solid. The material wasof sufficient purity to be used directly in the next step withoutfurther isolation and purification procedures. R_(f): ˜0.47(EtOAc/Hxn=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.41 (d, J=2.4 Hz, 1H),8.00 (dd, J=8.4, 2.4 Hz, 1H), 7.27 (d, J=9.0 Hz, 1H), 5.16 (q, J=6.3 Hz,1H), 2.43 (s, 3H), 1.96 (br. s, 1H), 1.50 (d, J=6.3 Hz, 3H) ppm. Theanalytical data correspond to the data of compound (5b) of Method A.

Step L: 2-(1-Bromoethyl)-1-methyl-4-nitro-benzene (51)

Following the General Procedure of Description 9,2-(1-bromoethyl)-1-methyl-4-nitro-benzene (51) was prepared from1-(2-methyl-5-nitro-phenyl)ethanol (5k) (13.7 g, 75.5 mmol) and ˜1.0 Mhydrogen bromide in acetic acid (HBr/HOAc) (prepared from 33 wt-% HBr inHOAc (14.6 mL, 83.1 mmol) and glacial acetic acid (68 mL)). Afteraqueous work-up, 17.6 g (95% yield) of the title compound (5m) wereobtained as a brown-colored viscous oil that crystallized to a beigesolid upon standing at room temperature. The material was of sufficientpurity to be used directly and without additional isolation andpurification in the next step. R_(f): ˜0.76 (ethyl acetate(EtOAc)/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.40 (d, J=2.4 Hz,1H), 8.05 (dd, J=8.7, 2.4 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H), 5.36 (q,J=6.9 Hz, 1H), 2.51 (s, 3H), 2.13 (d, J=6.9 Hz, 3H) ppm. Analytical datacorrespond compound (5c) of Method A.

Step M: 2-(2-Methyl-5-nitro-phenyl)propanenitrile (5m)

Following the General Procedure of Description 10,2-(2-methyl-5-nitro-phenyl)propanenitrile (5m) was prepared from2-(1-bromoethyl)-1-methyl-4-nitro-benzene (51) (12.0 g, 49.3 mmol) andsodium cyanide (NaCN) (2.91 g, 52.2 mmol) in anhydrousN,N-dimethylformamide (DMF) (165 mL). Purification by silica gel columnchromatography with ethyl acetate (EtOAc)/hexane mixtures(EtOAc/hexane=1:4 v/v→3:7 v/v→1:1 v/v) afforded 11.3 g (82% yield) ofthe title compound (5m) as a reddish-brownish solid. R_(f): ˜0.38 (ethylacetate (EtOAc)/hexane=1:4 v/v). R_(f): ˜0.58 (ethyl acetate(EtOAc)/hexane=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.32 (d, J=2.4 Hz,1H), 8.11 (dd, J=8.4, 2.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 4.10 (q,J=7.2 Hz, 1H), 2.50 (s, 3H), 1.70 (d, J=7.2 Hz, 3H) ppm. The analyticaldata correspond to the data of compound (5d) of Method A.

Step N: tert-Butyl 3-cyano-3-(2-methyl-5-nitro-phenyl)butanoate (5n)

Following the General Procedure of Description 11 (Variant A),tert-butyl 3-cyano-3-(2-methyl-5-nitro-phenyl)butanoate (5n) wasprepared from 2-(2-methyl-5-nitro-phenyl)propanenitrile (5m) (4.56 g,24.0 mmol) by deprotonation with a commercial solution of lithiumdiisopropylamide (LDA) (1.8 M in heptane/THF/ethylbenzene) (13.8 mL,27.6 mmol) and subsequent alkylation with commercial, neat tert-butyl2-bromoacetate (BrCH₂CO₂tBu) (6.48 mL, 8.56 g, 43.9 mmol) in anhydroustetrahydrofuran (THF) (freshly distilled over sodium benzophenone ketylradical) (120 mL). Purification by silica gel column chromatographyusing ethyl acetate (EtOAc)/hexane mixtures (EtOAc/hexane=1:4 v/v→1:3v/v) afforded 3.43 g (47% yield) of the title compound (5n) a yellowoil. R_(f): ˜0.49 (ethyl acetate (EtOAc)/hexane=1:4 v/v). R_(f): ˜0.75(ethyl acetate (EtOAc)/hexane=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.20(d, J=2.7 Hz, 1H), 8.10 (dd, J=8.4, 2.4 Hz, 1H), 7.42 (d, J=8.4 Hz, 1H),3.05 (d, J=15.3 Hz, 1H), 2.96 (d, J=15.3 Hz, 1H), 2.78 (s, 3H), 1.98 (s,3H), 1.34 (s, 9H) ppm.

Step O: tert-Butyl 3-(5-amino-2-methyl-phenyl)-3-cyano-butanoate (5o)

Following the General Procedure of Description 12, tert-butyl3-(5-amino-2-methyl-phenyl)-3-cyano-butanoate (5o) was prepared byreduction of tert-butyl 3-cyano-3-(2-methyl-5-nitro-phenyl)butanoate(5n) (5.89 g, 19.4 mmol) with nickel acetate tetrahydrate(Ni(OAc)₂.4H₂O) (964 mg, 3.87 mmol) and sodium borohydride (NaBH₄) (2.93g, 77.5 mmol) in a mixture of acetonitrile (MeCN) (55 mL) and water (5.5mL). Purification by silica gel column chromatography using a mixture ofethyl acetate (EtOAc) and hexane (EtOAc/hexane=1:2 v/v) afforded 5.23 g(98% yield) of the target compound (5o) as a viscous yellow oil. R_(f):˜0.40 (EtOAc/hexane=1:2 v/v). R_(f): ˜0.20 (EtOAc/hexane=1:4 v/v). ¹HNMR (300 MHz, CDCl₃): δ 6.97 (d, J=8.1 Hz, 1H), 6.70 (d, J=2.1 Hz, 1H),6.56 (dd, J=8.1, 2.1 Hz, 1H), 3.60 br. s, 2H), 2.97 (d, J=14.7 Hz, 1H),2.81 (d, J=15.3 Hz, 1H), 2.49 (s, 3H), 1.88 (s, 3H), 1.35 (s, 9H) ppm.MS (ESI+): m/z=275.25 (M+H⁺)⁺. (549.35 (2M+H⁺)⁺.

Step P: tert-Butyl3-(5-benzyloxycarbonylamino-2-methyl-phenyl)-3-cyano-butanoate (5p)

Following the General Procedure of Description 13, tert-butyl3-(5-benzyloxycarbonylamino-2-methyl-phenyl)-3-cyano-butanoate (5p) wasprepared from tert-butyl 3-(5-amino-2-methyl-phenyl)-3-cyano-butanoate(5o) (5.23 g, 19.1 mmol), and CbzCl) (3.16 mL, 3.58 g (3.77 g of 95 wt-%purity material), 21.0 mmol), and freshly powdered sodiumhydrogencarbonate (NaHCO₃) (1.76 g, 21.0 mmol) in tetrahydrofuran (THF)(90 mL). Purification by silica gel column chromatography using an ethylacetate (EtOAc)/hexane mixture (EtOAc/hexane=1:3 v/v) afforded 6.86 g(88% yield) of the title compound (5p) as a viscous pale yellow tocolorless oil. R_(f): ˜0.43 (EtOAc/hexane=1:4 v/v). R_(f): ˜0.75(EtOAc/hexane=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.48-7.30 (m, 6H),7.26-7.20 (m, 1H), 7.14 (d, J=8.1 Hz, 1H), 6.69 (br. s, 1H), 5.20 (s,2H), 2.99 (d, J=15.0 Hz, 1H), 2.99 (d, J=15.0 Hz, 1H), 2.82 (d, J=15.3Hz, 1H), 2.58 (s, 3H), 1.90 (s, 3H), 1.34 (s, 9H) ppm. MS (ESI+):m/z=431.15 (M+H⁺)⁺.

Step Q: tert-Butyl3-(5-benzyloxycarbonylamino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5q)

Following the General Procedure of Description 14, tert-butyl3-(5-benzyloxycarbonylamino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5q) was prepared by reduction of tert-butyl3-(5-benzyloxycarbonylamino-2-methyl-phenyl)-3-cyano-butanoate (5p)(6.64 g, 16.3 mmol) with nickel(II) chloride hexahydrate (NiCl₂.6H₂O)(1.93 g, 8.13 mmol) and sodium borohydride (NaBH₄) (4.68 g, 123.6 mmol)in the presence of di-tert-butyl-dicarbonate (Boc-anhydride, Boc₂O)(7.27 g, 33.3 mmol) in methanol (MeOH) (140 mL). Aqueous work-upfollowed repeated titruation of the crude powdered material overnightwith hexane (2×50 mL (removal of excess Boc₂O) yielded 6.66 g of acolorless powder after filtration, washing, and drying under reducedpressure. ¹H NMR analysis (300 MHz, CDCl₃) of the isolated materialshowed that the material consisted of a mixture of the title compound(5q) (82 mol-%, 5.53 g (66% yield)) and the side product tert-butyl4-(tert-butoxycarbonylamino)-3-[5-(tert-butoxycarbonylamino)-2-methyl-phenyl]-3-methyl-butanoate(5q′) (18 mol-%, 1.13 g). The combined filtrates were evaporated underreduced pressure. The residue was purified by silica gel columnchromatography using ethyl acetate (EtOAc)/hexane mixtures(EtOAc/hexane=1:5 v/v→1:4 v/v) to yield 870 mg of a colorless viscousoil. ¹H NMR analysis (300 MHz, CDCl₃) of the material purified by silicagel chromatography showed that the material consisted of a mixture ofthe title compound (5q) (44 mol-%, 397 mg (5% yield)) and the sideproduct (5q′) (56 mol-%, 641 mg) (5.93 g, 71% total yield). R_(f) (5q)˜0.41 (EtOAc/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃, (5q)): δ 7.42-7.28(m, 6H), 7.18 (d, J=2.1 Hz, 1H), 7.07 (d, J=8.4 Hz, 1H), 6.63 (br. s,1H), 5.19 (s, 2H), 4.60-4.50 (br. m, 1H), 3.63 (dd, J=13.8, 7.8 Hz, 1H),3.46 (dd, J=13.8, 4.8 Hz, 1H), 2.90 (d, J=14.1 Hz, 1H), 2.52 (s, 3H,partially superimposed), 2.51 (d, J=14.1 Hz, 1H, partiallysuperimposed), 1.61 (s, 3H), 1.40 (s, 9H), 1.19 (s, 9H) ppm. MS (ESI+,(5q)): m/z=535.25 (M+Na⁺)⁺. R_(f) (5q′) ˜0.50 (EtOAc/hexane=1:4 v/v).R_(f) (5q′): ˜0.75 (EtOAc/hexane=1:2 v/v). ¹H NMR (300 MHz, CDCl₃,(5q′)): δ 7.30 (br. d, J=7.5 Hz, 1H), 7.09 (d, J=2.4 Hz, 1H), 7.04 (d,J=8.4 Hz, 1H), 6.41 (br. s, 1H), 4.60-4.50 (br. m, 1H), 3.62 (dd,J=13.5, 7.8 Hz, 1H), 3.47 (dd, J=13.8, 4.8 Hz, 1H), 2.89 (d, J=14.1 Hz,1H), 2.51 (d, J=14.1 Hz, 1H, partially superimposed), 2.50 (s, 3H), 1.54(s, 3H), 1.51 (s, 9H), 1.41 (s, 9H), 1.20 (s, 9H) ppm. MS (ESI+, (5q′)):m/z=501.25 (M+Na⁺)⁺.

Step R: tert-Butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5r)

Following the General Procedure of Description 16, tert-butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5r) was prepared by hydrogenolysis of tert-butyl3-(5-benzyloxycarbonylamino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5q) (6.66 g of a mixture of (5q) (5.53 g, 10.8 mmol) and (5q′) (10.8mmol)) in the presence of 10 wt-% palladium on charcoal (Pd/C)containing −50 wt-% water (2.20 g) in a mixture of methanol (MeOH) (50mL) and ethyl acetate (EtOAc) (20 mL) and under an atmosphere ofhydrogen (˜15 psi, H₂-balloon). Purification by silica gel columnchromatography using ethyl acetate (EtOAc)/hexane mixtures(EtOAc/hexane=1:3 v/v→1:2 v/v) afforded 3.92 g (80% yield) of the titlecompound (5r) as a colorless very viscous oil and 1.03 g of the sideproduct (5q′) as a colorless solid. R_(f): ˜0.42 (EtOAc/hexane=1:2 v/v).R_(f): ˜0.14 (EtOAc/hexane=1:4 v/v). NMR (300 MHz, CDCl₃): δ 6.89 (d,J=7.8 Hz, 1H), 6.64 (d, J=1.8 Hz, 1H), 6.49 (dd, J=8.1, 2.7 Hz, 1H),4.60-4.46 (br. m, 1H), 3.60 (dd, J=13.5, 7.8 Hz, 1H, partiallysuperimposed), 3.51 (br. s, 2H, partially superimposed), 3.47 (dd,J=13.8, 4.5 Hz, 1H, partially superimposed), 2.86 (d, J=13.5 Hz, 1H),2.49 (d, J=14.1 Hz, 1H), 2.44 (s, 3H), 1.52 (s, 3H), 1.41 (s, 9H) 1.22(s, 9H) ppm. MS (ESI+): m/z=379.25 (M+H⁺)⁺, 401.20 (M+Na⁺)⁺.

Step S: tert-Butyl3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5s)

Following the General Procedure of Description 17 (Variant C),tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5s) was prepared from tert-butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5r) (730 mg, 1.93 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (1.27 mL, 10.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (503 mg,8.0 mmol) in a mixture of methanol (MeOH) (12 mL) and 85 wt-% phosphoricacid (H₃PO₄) (6 mL). Purification by silica gel column chromatographywith a mixture of ethyl acetate (EtOAc) and hexane (EtOAc/hexane=1:6v/v) afforded 735 mg (76% yield) of the title compound (5s) as an almostcolorless viscous oil that solidified to a slightly beige solid uponstanding at room temperature. R_(f): ˜0.56 (EtOAc/Hexane=1:4 v/v).R_(f): ˜0.73 (EtOAc/Hexane=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 6.99 (d,J=8.7 Hz, 1H), 6.63 (d, J=2.4 Hz, 1H), 6.50 (dd, J=8.7, 3.0 Hz, 1H),4.64-4.50 (br. m, 1H), 3.76-3.56 (m, 8H), 3.45 (dd, J=13.8, 4.5 Hz, 1H),2.90 (d, J=14.1 Hz, 1H), 2.51 (d, J=14.1 Hz, 1H), 2.46 (s, 3H), 1.55 (s,3H), 1.41 (s, 9H, 1.20 (s, 9H) ppm. MS (ESI+): m/z=503.20 (M+H⁺)⁺,525.20 (M+Na⁺)⁺.

Step T:4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (5)

Following the General Procedure of Description 23 (Variant A),4-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (5) was prepared from tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5s) (105 mg, 0.22 mmol) in a mixture of dichloromethane (DCM) (2.3 mL)and trifluoroacetic acid (TFA) (1.1 mL). Half of the solution (1.2 mL)was aliquoted off to yield 90 mg of the target compound (5) as a redoily to semi-solid di-trifluoroacetate salt after evaporation ofsolvents and final lyophilization from an aqueous solution. LC/UV (fromLC/MS): R_(t)=1.07 min. 96.1% purity by AUC at λ=254 nm. 1H NMR (300MHz, CD₃OD): δ 7.09 (d, J=8.7 Hz, 1H), 6.68 (d, J=2.7 Hz, 1H), 6.65 (dd,J=8.1, 2.7 Hz, 1H), 3.80-3.72 (m, 4H), 3.70-3.63 (m, 4H), 3.58 (d,J=13.5 Hz, 1H), 3.42 (d, J=13.5 Hz, 1H), 2.92 (s, 2H), 2.45 (3H), 1.68(s, 3H) ppm. MS (ESI+): m/z=347.10 (M+H+)+.

Following the General Procedure of Description 23 (Variant B),4-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (5) was prepared from tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(5s) (398 mg, 0.79 mmol) in 2 N HCl in diethyl ether (2.0 N HCl in Et₂O)(10 mL, 20 mmol) to yield 274 mg (82% recovery) of the target compound(5) as a slightly brownish powdery solid dihydrochloride salt afterevaporation of the solvents and lyophilization from an aqueous solution.The material was of sufficient purity to be used directly and withoutfurther isolation and purification procedures in in vitro and in vivoevaluation. LC/UV (from LC/MS): Rt=1.15 min. 96.1% purity by AUC atλ=254 nm. LC/UV: Rt=7.33 min. 96.3% purity by AUC at λ=254 nm. 1H NMR(300 MHz, CD₃OD): δ 7.21 (d, J=8.4 Hz, 1H), 7.02-6.96 (br. d, 1H), 6.92(br. d, J=8.1 Hz, 1H), 3.91-3.82 (br. m, 4H), 3.71-3.64 (br. m, 4H),3.60 (d, J=13.5 Hz, 1H), 3.42 (d, J=13.5 Hz, 1H), 3.01 (d, J=16.2 Hz,1H), 2.90 (d, J=16.2 Hz, 1H), 2.51 (s, 3H), 1.72 (s, 3H) ppm. MS (ESI+):m/z=347.10 (M+H+)+. (ESI−): m/z=345.05 (M−H+)−, 693.30 (2M−H+)−.

Example 64-Amino-3-[3-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (6)

Step A: 1-(2-Methyl-3-nitro-phenyl)ethanone (6a)

Following the General Procedure of Description 1 (Variant A),1-(2-methyl-3-nitro-phenyl)ethanone (6a) was prepared commercial1-(o-tolyl)ethanone (2′-methylacetophenone) (2.02 g, 15.0 mmol) in amixture of glacial acetic acid (HOAc) (10 mL) and white fuming nitricacid (min. 90 wt-% HNO₃) (20 mL). After aqueous work-up, the crudematerial consisted mainly of a mixture of two regioisomers1-(2-methyl-3-nitro-phenyl)ethanone (6a) and1-(2-methyl-5-nitro-phenyl)ethanone (6a′) and which were separated bysilica gel column chromatography using a mixture of ethyl acetate(EtOAc) and hexane (EtOAc/hexane=1:9) as an eluent to afford 1.17 g (44%yield) the title compound (6a) and 1.10 g (41% yield) of thecorresponding regioisomer (6a′) (1-(2-methyl-5-nitro-phenyl)ethanone) asa yellow solid. R_(f) (6a): ˜0.34 (ethyl acetate (EtOAc)/hexane=1:4v/v). R_(f)(6a): ˜0.16 (ethyl acetate (EtOAc)/hexane=1:9 v/v). ¹H NMR(300 MHz, CDCl₃, (6a)): δ 7.84 (dd, J=7.8, 1.2 H, 1H), 7.72 (dd, J=7.5,1.2 Hz, 1H), 7.46-7.38 (m, 1H), 2.61 (s, 3H), 2.52 (s, 3H) ppm.R_(f)(6a′): ˜0.51 (ethyl acetate (EtOAc)/hexane=1:4 v/v). R_(f)(6a′):˜0.25 (ethyl acetate (EtOAc)/hexane=1:9 v/v). ¹H NMR (300 MHz, CDCl₃,(6a′)): δ 8.55 (d, J=2.4 Hz, 1H), 8.22 (dd, J=8.7, 2.7 Hz, 1H), 7.44 (d,J=8.7 Hz, 1H), 2.67 (s, 3H), 2.63 (s, 3H) ppm.

Step B: 1-(2-Methyl-3-nitro-phenyl)ethanol (6b)

Following the General Procedure of Description 5,1-(2-methyl-3-nitro-phenyl)ethanol (6b) was prepared from1-(2-methyl-3-nitro-phenyl)ethanone (6a) (1.47 g, 8.22 mmol) and sodiumborohydride (NaBH₄) (156 mg, 4.11 mmol) in methanol (MeOH) (20 mL).After aqueous work-up, 1.52 g (˜quantitative % yield) of the titlecompound (6b) was obtained as a yellow viscous oil. The material was ofsufficient purity to be used directly and without additional isolationand purification in the next step. R_(f): ˜0.47 (ethyl acetate(EtOAc)/hexane=1:2 v/v); R_(f): ˜0.17 (ethyl acetate (EtOAc)/hexane=1:4v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.81 (dd, J=7.8, 0.9 Hz, 1H), 7.64 (dd,J=8.1, 1.5 Hz, 1H), 7.36 (t, J=7.8 Hz, 1H), 5.22 (br. q, J=6.3 Hz, 1H),2.41 (s, 3H), 1.91 (br. s, 1H), 1.48 (d, J=6.6 Hz) ppm.

Step C: 1-(1-Bromoethyl)-2-methyl-3-nitro-benzene (6c)

Following the General Procedure of Description 9,1-(1-bromoethyl)-2-methyl-3-nitro-benzene (6c) was prepared from1-(2-methyl-3-nitro-phenyl)ethanol (6b) (1.51 g, 8.35 mmol) and -1.0 Mhydrogen bromide in acetic acid (HBr/HOAc) (prepared from 33 wt-% HBr inHOAc (1.60 mL, 9.14 mmol) and glacial acetic acid (7.5 mL)). Afteraqueous work-up, 1.88 g (92% yield) of the title compound (6c) wereobtained as a brown-colored viscous oil. The material was of sufficientpurity to be used directly and without additional isolation andpurification in the next step. R_(f): ˜0.64 (ethyl acetate(EtOAc)/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.79 (dd, J=8.1, 1.2Hz, 1H), 7.67 (dd, J=8.1, 1.2 Hz, 1H), 7.37 (t, J=7.8 Hz, 1H), 5.41 (q,J=6.9 Hz, 1H), 2.48 (s, 3H), 2.09 (d, J=7.2 Hz, 3H) ppm.

Step D: 2-(2-Methyl-3-nitro-phenyl)propanenitrile (6d)

Following the General Procedure for the Preparation of Nitriles ofDescription 10, 2-(2-ethyl-3-nitro-phenyl)propanenitrile (6d) wasprepared from 1-(1-bromoethyl)-2-methyl-3-nitro-benzene (6c) and sodiumcyanide (NaCN) (451 mg, 9.18 mmol) in anhydrous N,N-dimethylformamide(DMF) (25 mL). Purification by silica gel column chromatography withethyl acetate (EtOAc)/hexane mixtures (EtOAc/hexane=1:4→1:3 v/v)afforded 1.04 g (72% yield) of the title compound (6d) as a yellowliquid. R_(f): ˜0.36 (ethyl acetate (EtOAc)/hexane=1:4 v/v). R_(f):˜0.61 (ethyl acetate (EtOAc)/hexane=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ7.75 (d, J=2.4 Hz, 1H), 7.72 (d, J=3.0 Hz, 1H), 7.42 (t, J=7.8 Hz, 1H),4.17 (q, J=7.5 Hz, 1H), 2.45 (s, 3H), 1.66 (d, J=7.5 Hz, 3H) ppm.

Step E: Ethyl 3-cyano-3-(2-methyl-3-nitro-phenyl)butanoate (6e)

Following the General Procedure of Description 11 (Variant A), ethyl3-cyano-3-(2-methyl-3-nitro-phenyl)butanoate (6e) was prepared from2-(2-ethyl-3-nitro-phenyl)propanenitrile (6d) (761 mg, 4.00 mmol) bydeprotonation with a commercial solution of lithium diisopropylamide(LDA) (1.8 M in heptane/THF/ethylbenzene) (2.56 mL, 4.60 mmol) andsubsequent alkylation with commercial, neat ethyl 2-bromoacetate(BrCH₂CO₂Et) (885 μL, 1.34 g, 8.00 mmol; dried over 4 Å molecular sieves(4 Å MS) prior to use) in anhydrous tetrahydrofuran (THF) (freshlydistilled over sodium benzophenone ketyl radical) (20 mL). Purificationby silica gel column chromatography using an ethyl acetate(EtOAc)/hexane mixture (EtOAc/hexane=1:4 v/v→1:3 v/v) afforded 562 mg(75% yield based on recovered starting material) of the title compound(6e) a yellow oil. R_(f): ˜0.43 (ethyl acetate (EtOAc)/hexane=1:2 v/v).R_(f): ˜0.18 (ethyl acetate (EtOAc)/hexane=1:4 v/v). ¹H NMR (300 MHz,CDCl₃): δ 7.65 (dd, J=8.1, 1.2 Hz, 1H, superimposed), 7.56 (dd, J=8.1,1.2 Hz, 1H, superimposed), 7.37 (t, J=8.4 Hz, 1H), 4.10 (q, J=6.9 Hz,2H), 3.11 (d, J=15.6 Hz, 1H), 3.00 (d, J=15.6 Hz, 1H), 2.69 (s, 3H),2.02 (s, 3H), 1.17 (t, J=7.2 Hz, 3H) ppm.

Step F: 4-β-Amino-2-methyl-phenyl)-4-methyl-pyrrolidin-2-one (6f)

Following the General Procedure of Description 4,4-β-amino-2-methyl-phenyl)-4-methyl-pyrrolidin-2-one (6f) was preparedfrom ethyl 3-cyano-3-(2-methyl-3-nitro-phenyl)butanoate (6e) (562 mg,2.03 mmol), freshly washed active Raney®-3202 nickel (about 10 mL ofslurry) in ethanol (EtOH) (80 mL) using a Parr hydrogenation apparatusunder about 60 psi hydrogen pressure. ¹H NMR analysis (300 MHz, CDCl₃)and TLC analysis showed that the reaction mixture consisted of a mixtureof completely reduced non-cyclized starting material ethyl4-amino-3-β-amino-2-methyl-phenyl)-3-methyl-butanoate (6f) and the titlecompound 4-β-amino-2-methyl-phenyl)-4-methyl-pyrrolidin-2-one (6f). Aconcentrated filtration alcoholic solution (about 20 mL) of the crudereaction mixture was stirred for about 2 hours at about 60° C. to 80° C.to facilitate lactamization of non-cyclized reduction product (6f′) tothe title compound (6f) together with slow evaporation of residualsolvent. The material thus obtained was further purified by silica gelcolumn chromatography using dichloromethane (DCM) and methanol (MeOH)mixtures (DCM/MeOH=96:4 v/v) as eluents to afford 221 mg (53% yield) ofthe target compound (6f) as a colorless solid. R_(f): ˜0.68(DCM/MeOH=9:1 v/v). R_(f): ˜0.23 (DCM/MeOH=95:5 v/v). ¹H NMR (300 MHz,CDCl₃): δ 7.01 (t, J=6.6 Hz, 1H), 6.64 (dd, J=7.5, 0.6 Hz, 1H), 6.57(dd, J=7.8, 0.6 Hz, 1H), 6.30-6.20 (br. m, 1H), 3.77 (br. d, J=9.3 Hz,1H), 3.64 (br. s, 2H, superimposed), 3.61 (br. dd, J=9.3, 1.5 Hz, 1H,superimposed), 2.86 (d, J=16.2 Hz, 1H), 2.58 (d, J=16.2 Hz, 1H), 2.14(s, 3H), 1.53 (s, 3H) ppm. MS (ESI+): m/z=205.20 (M+H⁺)⁺, 409.3(2M+H⁺)⁺.

Step G:4-[3-[Bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(6g)

Following the General Procedure of Description 17 (Variant A),4-[3-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(6g) was prepared from4-β-amino-2-methyl-phenyl)-4-methyl-pyrrolidin-2-one (6f) (221 mg, 1.08mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (695 μL, 5.48mmol), and sodium cyanoborohydride (NaBH₃CN) (275 mg, 4.38 mmol) in amixture of methanol (MeOH) (4.0 mL) and trifluoroacetic acid (TFA) (2.0mL). Aqueous work afforded ˜455 mg (˜quantitative yield) of crudematerial (6g) as a colorless viscous oil which was of sufficient purity(only residual ethyl acetate (EtOAc) and dichloromethane (DCM)) to beused directly and without further purification and isolation proceduresin the next step. R_(f): ˜0.31 (EtOAc). R_(f): ˜0.33 (DCM)/MeOH=95:5v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.16 (t, J=7.8 Hz, 1H, superimposed),7.09 (dd, J=8.1, 1.5 Hz, 1H), 6.88 (dd, J=7.8, 1.5 Hz, 1H), 6.46-6.38(br. m, 1H), 3.78 (br. d, J=9.9 Hz, 1H), 3.68 (br. dd, J=8.4, 1.2 Hz,1H), 3.50-3.34, m, 8H), 2.86 (d, J=16.2 Hz, 1H), 2.63 (d, J=16.2 Hz,1H), 2.36 (s, 3H), 1.46 (s, 3H) ppm. MS (ESI+): m/z=329.15 (M+H⁺)⁺.

Step H:4-Amino-3-[3-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (6)

Following the General Procedure of Description 22,4-amino-3-[3-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (6) was prepared from4-[3-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(6g) (˜355 mg, ˜1.08 mmol) by hydrolysis in concentrated hydrochloricacid (HCl) (about 15 mL) at reflux temperature for about 24 hours toafford a mixture containing the title compound (6) (minor) as adihydrochloride salt and the starting material (6g) (major) as ahydrochloride salt after isolation using evaporation and lyophilization.The material thus obtained was purified by preparative RP-HPLC using awater/acetonitrile/0.1 vol-% formic acid gradient to afford 25 mg of thetitle compound (6) as a slightly brown dihydrochloride salt after finallyophilization of the solvents in the presence of an excess of 1.0 Mhydrochloric acid (HCl). The material was of sufficient purity to beused directly and without further isolation and purification proceduresin in vitro and/or in vivo evaluation. LC/UV (from LC/MS): R_(t)=1.28min. Purity 98.4% by AUC at λ=254 nm. MS (ESI+): m/z=347.15 (M+H⁺)⁺.(ESI−): m/z=345.05 (M−H⁺)⁻, 693.30 (2M−H+)⁻.

Example 74-Amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (7) Method A

Step A: 2-Methyl-4-nitro-phenyl)methanol (7a)

Following the General Procedure of Description 6,2-methyl-4-nitro-phenyl)methanol (7a) was prepared from commercial2-methyl-4-nitro benzoic acid (5.0 g, 27.6 mmol) with boranedimethylsulfide complex (2.0 M BH₃. SMe₂ in THF) (27.6 mL, 55.2 mmol) inanhydrous tetrahydrofuran (100 mL) to yield 4.62 g (quantitative yield)of the target compound (7a) as a pale yellow solid, which was ofsufficient purity to be used directly in the next step without furtherisolation and purification. R_(f): ˜0.50 (EtOAc/Hxn=1:1 v/v). ¹H NMR(300 MHz, CDCl₃): δ 8.07 (dd, J=8.4, 2.1 Hz, 1H), 8.02 (d, J=2.1 Hz,1H), 7.62 (d, J=8.1 Hz, 1H), 4.79 (s, 2H), 2.38 (s, 3H), 1.87 (br. s,1H) ppm.

Step B: 2-Methyl-4-nitro-benzaldehyde (7b)

Following the General Procedure of Description 7 (Variant A),2-methyl-4-nitro-benzaldehyde (7b) was prepared from2-methyl-4-nitro-phenyl)methanol (7a) (1.63 g, 9.72 mmol) in thepresence of dimethylsulfoxide (DMSO) (5.68 mL, 6.25 g, 80.0 mmol),triethylamine (TEA, Et₃N) (6.95 mL, 5.05 g, 50.0 mmol), and sulfurtrioxide pyridine complex (SO₃-pyridine) (4.78 g, 30.0 mmol) inanhydrous dichloromethane (60 mL). Aqueous work-up afforded 1.51 g (94%yield) of the target compound (7b) as a yellow solid. The material wasof sufficient purity to be used directly in the next step withoutfurther isolation and purification procedures.

Following the General Procedure of Description 7 (Variant B),2-methyl-4-nitro-benzaldehyde (7b) was prepared from2-methyl-4-nitro-phenyl)methanol (7a) (8.4 g, 50.3 mmol) in the presenceof manganese dioxide (MnO₂) (48.1 g, 553 mmol). Work-up afforded 7.5 g(90% yield) of the target compound (7b) as a yellow solid. The materialwas of sufficient purity to be used directly in the next step withoutfurther isolation and purification procedures. R_(f): ˜0.58(EtOAc/Hxn=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 10.39 (s, 1H), 8.20 (dd,J=8.4, 2.1 Hz, 1H), 8.14 (br. s, 1H), 7.98 (d, J=8.1 Hz, 1H), 2.79 (s,3H) ppm. The compound is also commercially available.

Step C: 1-(2-Methyl-4-nitro-phenyl)ethanol (7c)

Following the General Procedure of Description 8, MeTiCl₃ was freshlyprepared prior to use from commercial titanium tetrachloride (TiCl₄)(4.73 mL, 8.17 g, 43.1 mmol) and methyl lithium (MeLi) (1.6 M in Et₂O,26.9 mL, 43.0 mmol) in anhydrous diethyl ether (Et₂O) (230 mL). Asolution of 2-methyl-4-nitro-benzaldehyde (7b) (7.11 g, 43.0 mmol) inanhydrous Et₂O (200 mL) was added. Aqueous work-up afforded 7.0 g (90%yield) of the target compound (7c) as a yellow solid. The material wasof sufficient purity to be used directly in the next step withoutfurther isolation and purification procedures. R_(f): ˜0.24(EtOAc/Hxn=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.07 (dd, J=8.7, 2.7 Hz,1H), 7.99 (d, J=2.4 Hz, 1H), 7.72 (d, J=9.0 Hz, 1H), 5.18 (q, J=6.6 Hz,1H), 2.42, (s, 3H), 1.92-1.88 (br. m, 1H), 1.47 (d, J=6.3 Hz, 3H) ppm.

Step D: 1-(1-Bromoethyl)-2-methyl-4-nitro-benzene (7d)

Following the General Procedure of Description 9,1-(1-bromoethyl)-2-methyl-4-nitro-benzene (7d) was prepared from1-(2-methyl-4-nitro-phenyl)ethanol (7c) (7.05 g, 38.9 mmol) and -1.0 Mhydrogen bromide in acetic acid (HBr/HOAc) (prepared from 33 wt-% HBr inHOAc (7.5 mL, 42.8 mmol) and glacial acetic acid (35.0 mL)). Afteraqueous work-up, 9.0 g (95% yield) of the title compound (7d) wereobtained as a brown-colored viscous oil. The material was of sufficientpurity to be used directly and without additional isolation andpurification in the next step. R_(f): ˜0.67 (ethyl acetate(EtOAc)/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.08 (dd, J=8.7, 2.1Hz, 1H), 8.03 (d, J=2.1 Hz, 1H), 7.70 (d, J=8.7 Hz, 1H), 5.35 (q, J=6.9Hz, 1H), 2.51 (s, 3H), 2.09 (d, J=6.9 Hz) ppm.

Step E: 2-(2-Methyl-4-nitro-phenyl)propanenitrile (7e)

Following the General Procedure of Description 10,2-(2-methyl-4-nitro-phenyl)propanenitrile (7e) was prepared from1-(1-bromoethyl)-2-methyl-4-nitro-benzene (7d) (9.0 g, 36.9 mmol) andsodium cyanide (NaCN) (2.2 g, 44.3 mmol) in anhydrousN,N-dimethylformamide (DMF) (70 mL). Purification by silica gel columnchromatography with ethyl acetate (EtOAc)/hexane mixtures(EtOAc/hexane=1:3 v/v→1:2 v/v) afforded 5.0 g (71% yield) of the titlecompound (7e) as a brown oil. R_(f): ˜0.45 (EtOAc/hexane=1:2 v/v).R_(f): ˜0.29 (EtOAc/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.13(dd, J=8.4, 2.4 Hz, 1H), 8.08 (d, J=2.7 Hz, 1H), 7.65 (d, J=8.1 Hz, 1H),4.12 (q, J=7.5 Hz, 1H), 2.48 (s, 3H), 1.65 (d, J=7.2 Hz) ppm.

Step F: Ethyl 3-cyano-3-(2-methyl-4-nitro-phenyl)butanoate (7f)

Following the General Procedure of Description 11 (Variant A), ethyl3-cyano-3-(2-methyl-4-nitro-phenyl)butanoate (7f) was prepared from2-(2-methyl-4-nitro-phenyl)propanenitrile (7e) (718 mg, 3.78 mmol) bydeprotonation with a commercial solution of lithium diisopropylamide(LDA) (1.8 M in heptane/THF/ethylbenzene) (2.50 mL, 4.50 mmol) andsubsequent alkylation with commercial, neat ethyl 2-bromoacetate(BrCH₂CO₂Et) (885 μL, 1.34 g, 8.00 mmol; dried over 4 Å molecular sieves(4 Å MS) prior to use) in anhydrous tetrahydrofuran (THF) (freshlydistilled over sodium benzophenone ketyl radical) (18 mL). Purificationby silica gel column chromatography using an ethyl acetate(EtOAc)/hexane mixture (EtOAc/hexane=1:4 v/v) afforded 798 mg of amixture of the target compound (7f) and the starting material (7e)corresponding to 486 mg (47% yield based on recovered starting material)of the title compound (7f) a viscous yellow oil. R_(f): ˜0.50 (ethylacetate (EtOAc)/hexane=1:2 v/v). R_(f): ˜0.21 (ethyl acetate(EtOAc)/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.08 (dd, J=8.4, 2.7Hz, 1H), 8.04 (d, J=2.4 Hz, 1H), 7.53 (d, J=8.7 Hz, 1H), 4.11 (q, J=6.9Hz, 2H), 3.14 (d, J=15.9 Hz, 1H), 3.03 (d, J=15.9 Hz, 1H), 2.77 (s, 3H),1.98 (s, 3H), 1.17 (t, J=7.2 Hz, 3H) ppm.

Step G: 4-(4-Amino-2-methyl-phenyl)-4-methyl-pyrrolidin-2-one (7g)

Following the General Procedure of Description 4,4-(4-amino-2-methyl-phenyl)-4-methyl-pyrrolidin-2-one (7g) was preparedfrom ethyl 3-cyano-3-(2-methyl-4-nitro-phenyl)butanoate (7f) (486 mg,2.02 mmol), freshly washed active Raney®-3202 nickel (about 8.0 mL ofslurry) in ethanol (EtOH) (80 mL) using a Parr hydrogenation apparatusunder about 60 psi hydrogen pressure. ¹H NMR analysis (300 MHz, CDCl₃)and TLC analysis showed that the reaction mixture consisted of a mixtureof completely reduced non-cyclized starting material ethyl4-amino-3-(4-amino-2-methyl-phenyl)-3-methyl-butanoate (7g′) and thetitle compound 4-(4-amino-2-methyl-phenyl)-4-methyl-pyrrolidin-2-one(7g). A concentrated filtration alcoholic solution (about 20 mL) of thecrude reaction mixture was stirred for about 2 hours at about 60-80° C.to facilitate lactamization of non-cyclized reduction product (7g′)(4-amino-3-(4-amino-2-methyl-phenyl)-3-methyl-butanoic acid) to thetitle compound (7g) together with slow evaporation of residual solvent.The material thus obtained was further purified by silica gel columnchromatography using a dichloromethane (DCM) and methanol (MeOH) mixture(DCM/MeOH=95:5 v/v) as eluents to afford 224 mg (54% yield) of thetarget compound (7g) as a colorless solid. R_(f): ˜0.59 (DCM/MeOH=9:1v/v). ¹H NMR (300 MHz, CDCl₃): δ 6.87-6.81 (m, 1H), 6.57-6.48 (m,superimposed signals, 2H), 6.11-6.05 (br. m, 1H), 3.72 (br. d, J=9.3 Hz,1H), 3.59 (br. s, 2H, superimposed), 3.57 (br. dd, J=9.3, 1.5 Hz, 1H,superimposed), 2.81 (d, J=16.2 Hz, 1H), 2.52 (d, J=16.2 Hz, 1H), 2.28(s, 3H), 1.45 (s, 3H) ppm. MS (ESI+): m/z=205.20 (M+H⁺)⁺, 409.3(2M+H⁺)⁺.

Step H:4-[4-[Bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(7h)

Following the General Procedure of Description 17 (Variant A),4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(7h) was prepared from4-(4-amino-2-methyl-phenyl)-4-methyl-pyrrolidin-2-one (7g) (224 mg, 1.10mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (695 μL, 5.48mmol), and sodium cyanoborohydride (NaBH₃CN) (275 mg, 4.38 mmol) in amixture of methanol (MeOH) (4.0 mL) and trifluoroacetic acid (TFA) (2.0mL). Aqueous work afforded ˜393 mg (˜quantitative yield) of crudematerial (7h) as a colorless viscous oil which was of sufficient purity(only residual ethyl acetate (EtOAc) and dichloromethane (DCM)) to beused directly and without further purification and isolation proceduresin the next step. R_(f): ˜0.22 (EtOAc). R_(f): ˜0.63 (DCM)/MeOH)=9:1v/v). ¹H NMR (300 MHz, CDCl₃): δ 6.94 (d, J=8.7 Hz, 1H), 6.50 (dd,J=8.7, 3.0 Hz, 1H), 6.45 (d, J=2.7 Hz, 1H), 6.17 (br. m, 1H), 3.76-3.56(br. m, 10H), 2.82 (d, J=16.2 Hz, 1H), 2.54 (d, J=16.2 Hz, 1H), 2.34 (s,3H), 1.46 (s, 3H) ppm. MS (ESI+): m/z=329.15 (M+H⁺)⁺.

Step I:4-Amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (7)

Following the General Procedure of Description 22,4-amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (7) was prepared from4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-methyl-pyrrolidin-2-one(7h) (˜361 mg, 1.10 mmol) by hydrolysis in concentrated hydrochloricacid (HCl) (about 15 mL) at reflux temperature for about 24 hours toyield the target compound (7) as a dihydrochloride salt and the startingmaterial (7h) (major) as a hydrochloride salt after isolation usingevaporation and lyophilization. Part of the material thus obtained waspurified by preparative RP-HPLC using a water/acetonitrile/0.1 vol-%formic acid gradient to afford 98 mg of the title compound (7) as acolorless foamy dihydrochloride salt after final lyophilization of thesolvents in the presence of an excess of 1.0 M hydrochloric acid (HCl).The material was of sufficient purity to be used directly and withoutfurther isolation and purification procedures in in vitro and/or in vivoevaluation. LC/UV (of LC/MS): R_(t)=0.89 min.>99% purity by AUC at λ=254nm. ¹H NMR (300 MHz, CD₃OD): δ 7.23 (d, J=9.3 Hz, 1H), 6.72-6.64 (m,2H), 3.84-3.74 (br. m, 4H), 3.70-3.62 (br. m, 4H), 3.56 (d, J=13.2 Hz,1H), 3.36 (d, J=13.2 Hz, 1H), 2.89 (s, 2H), 2.53 (s, 3H), 1.67 (s, 3H)ppm. MS (ESI+): m/z=347.10 (M+H⁺)⁺. (ESI−): m/z=345.05 (M−H⁺)⁻, 693.30(2M−H⁺)⁻.

Method B

Step J: tert-Butyl 3-cyano-3-(2-methyl-4-nitro-phenyl)butanoate (7j)

Following the General Procedure of Description 11 (Variant B),tert-butyl 3-cyano-3-(2-methyl-4-nitro-phenyl)butanoate (7j) wasprepared from 2-(2-methyl-4-nitro-phenyl)propanenitrile (7e) (preparedfrom commercial 2-methyl-4-nitro benzoic acid as described in stepsa)-e) for Method A) (2.18 g, 11.44 mmol) by deprotonation with sodiumhydride (504 mg of a 60 wt-% suspension in mineral oil, 302 mg, 12.6mmol) in a mixture of dimethylsulfoxide (DMSO) (28 mL) and diethylether(Et₂O) (41 mL) and subsequent alkylation with commercial, neattert-butyl 2-bromoacetate (BrCH₂CO₂tBu) (3.38 mL, 4.46 g, 22.89 mmol).Purification by silica gel column chromatography using an ethyl acetate(EtOAc)/hexane mixture (EtOAc/hexane=1:5 v/v) afforded 3.48 g (99%)yield) of the title compound (7j) as a viscous yellow oil. R_(f): ˜0.42(EtOAc/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.09 (d, J=2.1 Hz,1H), 8.05 (dd, J=9.0, 3.0 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H), 3.05 (d,J=15.3 Hz, 1H), 2.95 (d, J=15.3 Hz, 1H), 2.77 (s, 3H), 1.95 (s, 3H),1.33 (s, 9H) ppm.

Step K: tert-Butyl 3-(4-amino-2-methyl-phenyl)-3-cyano-butanoate (7k)

Following the General Procedure of Description 12, tert-butyl3-(4-amino-2-methyl-phenyl)-3-cyano-butanoate (7k) was prepared byreduction of tert-butyl 3-cyano-3-(2-methyl-4-nitro-phenyl)butanoate(7j) (3.51 g, 11.54 mmol) with nickel acetate tetrahydrate(Ni(OAc)₂.4H₂O) (574 mg, 2.31 mmol) and sodium borohydride (NaBH₄) (1.75g, 46.1 mmol) in a mixture of acetonitrile (MeCN) (30 mL) and water (3mL). Aqueous work-up afforded 3.23 g (˜quantitative yield) of the targetcompound (7k) as pale yellow oil. The material contained some residualorganic solvent but was of sufficient purity to be used directly andwithout further isolation or purification procedures in the next step.R_(f): ˜0.40 (EtOAc/hexane=1:2 v/v). R_(f): ˜0.14 (EtOAc/hexane=1:4v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.08 (d, J=8.4 Hz, 1H), 6.53 (d, J=2.4Hz, 1H), 6.49 (dd, J=8.4, 2.4 Hz, 1H), 3.64 (br. s, 2H), 2.94 (d, J=15.0Hz, 1H), 2.78 (d, J=15.0 Hz, 1H), 2.54 (s, 3H), 1.88 (s, 3H), 1.35 (s,9H) ppm. MS (ESI+): m/z=275.25 (M+H⁺)⁺. (549.35 (2M+H⁺)⁺.

Step L: tert-Butyl3-(4-benzyloxycarbonylamino-2-methyl-phenyl)-3-cyano-butanoate (71)

Following the General Procedure of Description 13, tert-butyl3-(4-benzyloxycarbonylamino-2-methyl-phenyl)-3-cyano-butanoate (71) wasprepared from tert-butyl 3-(4-amino-2-methyl-phenyl)-3-cyano-butanoate(7k) (3.32 g, 12.1 mmol), and CbzCl) (2.0 mL, 2.27 g (2.34 g of 95 wt-%purity material), 13.31 mmol), and freshly powdered sodiumhydrogencarbonate (NaHCO₃) (1.12 g, 13.31 mmol) in tetrahydrofuran (THF)(75 mL). Purification by silica gel column chromatography using an ethylacetate (EtOAc)/hexane mixture (EtOAc/hexane=1:4 v/v) afforded 3.86 g(78% yield) of the title compound (71) as a viscous yellow oil. R_(f):˜0.41 (EtOAc/hexane=1:4 v/v). R_(f): ˜0.66 (EtOAc/hexane=1:2 v/v). ¹HNMR (300 MHz, CDCl₃): δ 7.42-7.32 (m, 5H), 7.26-7.22 (m, 3H), 6.69 (br.s, 1H), 5.18 (s, 2H), 2.94 (d, J=15.0 Hz, 1H), 2.97 (d, J=14.7 Hz, 1H),2.83 (d, J=15.0 Hz, 1H), 2.61 (s, 3H), 1.90 (s, 3H), 1.34 (s, 9H) ppm.MS (ESI+): m/z=431.15 (M+H⁺)⁺, 549.35 (2M+H⁺)⁺.

Step M: tert-Butyl3-(4-benzyloxycarbonylamino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7m)

Following the General Procedure of Description 14, tert-butyl3-(4-benzyloxycarbonylamino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7m) was prepared by reduction of tert-butyl3-(4-benzyloxycarbonylamino-2-methyl-phenyl)-3-cyano-butanoate (71)(3.59 g, 8.88 mmol) with nickel(II) chloride hexahydrate (NiCl₂.6H₂O)(1.04 g, 4.39 mmol) and sodium borohydride (NaBH₄) (2.50 g, 66.0 mmol)in the presence of di-tert-butyl-dicarbonate (Boc-anhydride, Boc₂O)(3.88 g, 17.76 mmol) in methanol (MeOH) (80 mL). Aqueous work-upfollowed by purification by silica gel column chromatography using ethylacetate (EtOAc)/hexane mixtures (EtOAc/hexane=1:5 v/v→1:1 v/v→3:1 v/v)afforded 1.82 g (40%) yield) of the title compound (7m) as a colorlesssolid. R_(f): ˜0.36 (EtOAc/hexane=1:4 v/v). ¹H NMR (300 MHz, CDCl₃): δ7.44-7.30 (m, 5H), 7.24-7.10 (m, 3H), 6.60 (br. s, 1H), 5.19 (s, 2H),4.56-4.42 (br. m, 1H), 3.60 (dd, J=13.8, 7.8 Hz, 1H), 3.44 (dd, J=13.8,4.5 Hz, 1H), 2.89 (d, J=13.8 Hz, 1H), 2.54 (s, 3H), 2.50 (d, J=14.1 Hz,1H), 1.54 (s, 3H), 1.40 (s, 9H), 1.19 (s, 9H) ppm. MS (ESI+): m/z=535.25(M+Na⁺)⁺.

Step N: tert-Butyl3-(4-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7n)

Following the General Procedure of Description 16, tert-butyl3-(4-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7n) was prepared by hydrogenolysis of tert-butyl3-(4-benzyloxycarbonylamino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7m) (1.83 g, 3.56 mmol) in the presence of 10 wt-% palladium oncharcoal (Pd/C) containing ˜50 wt-% water (0.75 g) in a mixture ofmethanol (MeOH) (30 mL) and ethyl acetate (EtOAc) (30 mL) and under anatmosphere of hydrogen (˜15 psi, H₂-balloon). Purification by silica gelcolumn chromatography using ethyl acetate (EtOAc)/hexane mixtures(EtOAc/hexane=1:3 v/v→1:2 v/v) afforded 980 mg (73%) yield) of the titlecompound (7n) as a colorless very viscous oil. R_(f): ˜0.14(EtOAc/hexane=1:4 v/v). R_(f): ˜0.35 (EtOAc/hexane=1:2 v/v). NMR (300MHz, CDCl₃): δ 7.04 (d, J=8.4 Hz, 1H), 6.50-6.42 (m, 2H), 4.56-4.44 (br.m, 1H), 3.58 (dd, J=13.8, 8.1 Hz, 1H, partially superimposed), 3.54 (br.s, 2H, partially superimposed), 3.42 (dd, J=13.5, 4.5 Hz, 1H), 2.85 (d,J=13.5 Hz, 1H), 2.47 (d, J=13.8 Hz, 1H, partially superimposed), 2.47(s, 3H, partially superimposed), 1.51 (s, 3H), 1.40 (s, 9H) 1.21 (s, 9H)ppm. MS (ESI+): m/z=379.25 (M+H⁺)⁺, 401.20 (M+Na⁺)⁺.

Step O: tert-Butyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7o)

Following the General Procedure of Description 17 (Variant B),tert-butyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7o) was prepared from tert-butyl3-(4-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7n) (962 mg, 2.54 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (1.6 mL, 12.7 mmol), and sodium cyanoborohydride (NaBH₃CN) (638 mg,10.2 mmol) in a mixture of methanol (MeOH) (15 mL) and acetic acid(HOAc) (7.7 mL). Purification by silica gel column chromatography with amixture of ethyl acetate (EtOAc) and hexane (EtOAc/hexane=1:6 v/v)afforded 941 mg (74% yield) of the title compound (7o) as an almostcolorless viscous oil.

Following the General Procedure of Description 17 (Variant C),tert-butyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7o) was prepared from tert-butyl3-(4-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7n) (855 mg, 2.26 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (3.0 mL, 23.6 mmol), and sodium cyanoborohydride (NaBH₃CN) (1.14 g,18.2 mmol) in a mixture of methanol (MeOH) (14 mL) and 85 wt-%phosphoric acid (H₃PO₄) (7 mL). Purification by silica gel columnchromatography with a mixture of ethyl acetate (EtOAc) and hexane(EtOAc/hexane=1:6 v/v) afforded 706 mg (74% yield) of the title compound(7o) as an almost colorless viscous oil. R_(f): ˜0.47 (EtOAc/Hexane=1:4v/v). ¹H NMR (300 MHz, CDCl₃): δ 7.14 (d, J=9.0 Hz, 1H), 6.50-6.40 (m,2H), 4.60-4.5 (br. m, 1H), 3.76-3.66 (m, 4H), 3.66-3.54 (m, 5H), 3.44(dd, J=13.8, 4.8 Hz, 1H), 2.85 (d, J=13.8 Hz, 1H), 2.54 (s, 3H), 2.49(d, J=14.1 Hz, 1H), 1.52 (s, 3H), 1.41 (s, 9H), 1.20 (s, 9H) ppm. MS(ESI+): m/z=503.20 (M+H⁺)⁺, 525.20 (M+Na⁺)⁺.

Step P:4-Amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (7)

Following the General Procedure of Description 23 (Variant B),4-amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (7) was prepared from tert-butyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7o) (161 mg, 0.32 mmol) in 2 N HCl in diethyl ether (2 N HCl in Et₂O)(6 mL, 12 mmol) to yield 114 mg (83% recovery) of the target compound(7) as an off-white powdery solid dihydrochloride salt after evaporationof the solvents and lyophilization from an aqueous solution. Thematerial was of sufficient purity to be used directly and withoutfurther isolation and purification procedures in in vitro and/or in vivoevaluation. LC/UV: R_(t)=7.57 min. 98.2% purity by AUC at λ=254 nm. ¹HNMR (300 MHz, CD₃OD): δ 7.25 (d, J=9.6 Hz, 1H), 6.80-6.68 (m, 2H),3.85-3.77 (br. m, 4H), 3.70-3.63 (br. m, 4H), 3.57 (d, J=13.2 Hz, 1H),3.36 (d, J=13.2 Hz, 1H), 2.89 (s, 2H), 2.54 (s, 3H), 1.66 (s, 3H) ppm.MS (ESI+): m/z=347.10 (M+H⁺)⁺. (ESI−): m/z=345.05 (M−H⁺)⁻, 693.30(2M−H⁺)⁻.

Following the General Procedure of Description 23 (Variant A),4-amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (7) was prepared from tert-butyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7o) (1.07 g, 2.11 mmol) in a mixture of dichloromethane (DCM) (24 mL)and trifluoroacetic acid (TFA) (12 mL) to yield 1.60 g of the targetcompound (7) as a red oily di-trifluoroacetate salt after evaporation ofsolvents and final lyophilization from an aqueous solution. LC/UV (fromLC/MS): R_(t)=1.01 min. 95.7% purity by AUC at λ=254 nm. ¹H NMR (300MHz, CDCl₃): δ 7.05 (d, J=8.7 Hz, 1H), 6.62-6.55 (m, 2H), 3.80-3.68 (br.m, 4H), 3.66-3.56 (br. m, 4H), 3.25 (d, J=16.8 Hz, 1H), 2.81 (d, J=16.5Hz, 1H), 2.47 (s, 3H), 2.10 (s, 2H), 1.57 (s, 3H) ppm. MS (ESI+):m/z=347.10 (M+H⁺)⁺. The di-trifluoroacetate salt of the target compound(7) (˜195 mg, ˜0.34 mmol) material was partially converted to thedihydrochloride salt of (7) by repeated precipitation with 2 N HCl inEt₂O (2×4 mL, 2×8.0 mmol) followed by washing of the precipitate withanhydrous Et₂O (3×4 mL) to yield 98 mg (69% recovery) of the targetcompound (7) as an off-white powdery solid dihydrochloride salt afterfinal lyophilization from an 50 vol-% aqueous acetonitrile solution.

Example 84-Amino-3-[5-[bis(2-chloroethyl)carbamoyl]-2-methyl-phenyl]butanoic acid(8)

Step A: Methyl 3-[(E)-3-tert-butoxy-3-oxo-prop-1-enyl]-4-methyl-benzoate(8a)

Following the General Procedure of Description 2 (Variant A), methyl3-[(E)-3-tert-butoxy-3-oxo-prop-1-enyl]-4-methyl-benzoate (8a) isprepared from known methyl 3-formyl-4-methyl-benzoate (3.56 g, 20.0mmol), commercial tert-butyl diethyl phosphonoacetate (5.87 mL, 6.31 g,25.0 mmol), and anhydrous lithium bromide (LiBr) (2.61 g, 30.0 mmol) ina mixture of triethylamine (Et₃N, TEA) (3.83 mL, 2.78 g, 27.5 mmol) andacetonitrile (MeCN) (20 mL). Purification by silica gel columnchromatography with a mixture of ethyl acetate (EtOAc) and hexanefurnishes the title compound (8a).

Step B: Methyl3-[3-tert-butoxy-1-(nitromethyl)-3-oxo-propyl]-4-methyl-benzoate (8b)

Following the General Procedure of Description 3, methyl3-[3-tert-butoxy-1-(nitromethyl)-3-oxo-propyl]-4-methyl-benzoate (8b) isprepared from methyl3-[(E)-3-tert-butoxy-3-oxo-prop-1-enyl]-4-methyl-benzoate (8a) (4.14 g,15.0 mmol) in a mixture of acetonitrile (MeCN) (40 mL) and nitromethane(MeNO₂) (8.2 mL, 9.3 g, 151 mmol) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.27 mL, 2.31 g, 15.2 mmol).Purification by silica gel column chromatography with mixtures of ethylacetate (EtOAc) and hexane furnishes the title compound (8b).

Step C: Methyl3-[1-(aminomethyl)-3-tert-butoxy-3-oxo-propyl]-4-methyl-benzoate (8c)

Following the General Procedure of Description 16, methyl3-[1-(aminomethyl)-3-tert-butoxy-3-oxo-propyl]-4-methyl-benzoate (8c) isprepared by global reduction of methyl3-[3-tert-butoxy-1-(nitromethyl)-3-oxo-propyl]-4-methyl-benzoate (8b)(3.37 g, 10.0 mmol) in the presence of 10 wt-% palladium on charcoal(Pd/C) containing ˜50 wt-% water (˜1.5 g) in methanol (MeOH) (40 mL) andunder an atmosphere of hydrogen (˜15 psi, H₂-balloon). The crudematerial after filtration may be used directly and without furtherisolation in the next step or may be purified by preparative HPLC toafford the title compound (8c).

Step D: Methyl3-[3-tert-butoxy-1-[(tert-butoxycarbonylamino)methyl]-3-oxo-propyl]-4-methyl-benzoate(8d)

Following the General Procedure of Description 15, methyl3-[3-tert-butoxy-1-[(tert-butoxycarbonylamino)methyl]-3-oxo-propyl]-4-methyl-benzoate(8d) is prepared from methyl3-[1-(aminomethyl)-3-tert-butoxy-3-oxo-propyl]-4-methyl-benzoate (8c)(3.07 g, 10.0 mmol) in anhydrous dichloromethane (DCM) (30 mL) at about0° C. (ice bath) with di-tert-butyl dicarbonate (Boc₂O) (2.29 g, 10.5mmol) in the presence of triethylamine (Et₃N, TEA) (1.67 mL, 1.21 g,12.0 mmol) and a catalytic amount of 4-(N,N-dimethylamino)pyridine(DMAP) (61 mg, 0.5 mmol). Aqueous work-up and purification by silica gelcolumn chromatography with mixtures of ethyl acetate (EtOAc) and hexaneafford the title compound (8d).

Step E:3-[3-tert-Butoxy-1-[(tert-butoxycarbonylamino)methyl]-3-oxo-propyl]-4-methyl-benzoicacid (8e)

Adapting a literature known protocol (Dayal, et al., Steroids, 1990,55(5), 233-237), a reaction mixture of methyl3-[3-tert-butoxy-1-[(tert-butoxycarbonylamino)methyl]-3-oxo-propyl]-4-methyl-benzoate(8d) (4.08 g, 10.0 mmol) and commercial lithium hydroxide monohydrate(LiOH.H₂O) in a mixture of water (20 mL) and methanol (MeOH) (5 mL) isstirred at room temperature. The reaction is monitored by TLC and/orLC/MS to completion. The solvent is partially removed under reducedpressure using a rotary evaporator. The pH of the residue is adjusted to˜1-2 with a 1.0M hydrochloric acid (HCl). The aqueous phase is extractedwith ethyl acetate (4×) and the combined organic extracts are washedwith and brine, dried over anhydrous magnesium sulfate (MgSO₄),filtered, and the solvents removed under reduced pressure using a rotaryevaporator to yield the target compound3-[3-tert-butoxy-1-[(tert-butoxycarbonylamino)methyl]-3-oxo-propyl]-4-methyl-benzoicacid (8e), which can be used directly in the next step.

Step F: tert-Butyl3-[5-[bis(2-chloroethyl)carbamoyl]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(8f)

Adapting a literature known protocol (U.S. Pat. No. 3,235,594), to areaction mixture of3-[3-tert-butoxy-1-[(tert-butoxycarbonylamino)methyl]-3-oxo-propyl]-4-methyl-benzoicacid (8e) (787 mg, 2.0 mmol), N-hydroxysuccinimide (NHS, HOSu) (235 mg,2.2 mmol) in anhydrous acetonitrile (MeCN) (10 mL) is added soliddicyclohexylcarbodiimide (DCC) (433 mg, 2.1 mmol) in small portions atabout room temperature. The reaction mixture is stirred for about 12hours and the precipitated dicyclohexylurea side product is filtered offusing a Büchner funnel. The filtrate is treated with commercialdi-(2-chloroethyl)amine hydrochloride(2-chloro-N-(2-chloroethyl)ethanamine hydrochloride;HN(CH₂—CH₂—Cl)₂.HCl) (393 mg, 2.2 mmol) followed by neat triethylamine(Et₃N, TEA) (321 μL, 233 mg, 2.3 mmol). The reaction mixture is stirredfor about 12 hours at room temperature. The reaction is followed by TLCand/or LC/MS to completion. The volatile solvents are removed underreduced pressure using a rotary evaporator. The residue is diluted with1.0M hydrochloric acid (HCl). The aqueous phase is extracted with ethylacetate (EtOAc) and the combined organic extracts are washed with brine,dried over anhydrous magnesium sulfate (MgSO₄), filtered, and evaporatedto dryness. Purification by silica gel column chromatography using EtOAcand hexane mixtures furnishes the target compound (8f).

Step G:4-Amino-3-[5-[bis(2-chloroethyl)carbamoyl]-2-methyl-phenyl]butanoic acid(8)

Following the General Procedure of Description 23 (Variant B),4-amino-3-[5-[bis(2-chloroethyl)carbamoyl]-2-methyl-phenyl]butanoic acid(8) is prepared from tert-butyl3-[5-[bis(2-chloroethyl)carbamoyl]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-butanoate(8f) (518 mg, 1.0 mmol) in 2.0 N HCl in diethyl ether (2.0 N HCl inEt₂O) (10 mL, 20 mmol) to yield the target compound (8) as a solidhydrochloride salt after evaporation of the solvents and lyophilizationfrom an aqueous solution. The material may be further purified bypreparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent of 1.0 Mhydrochloric acid (HCl).

Example 94-Amino-3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (9)

Step A: tert-Butyl (E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (9a)

Following the General Procedure of Description 2 (Variant A), tert-butyl(E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (9a) is prepared fromcommercial 2-methyl-5-nitro-benzaldehyde (5j) (for a synthesis of (5j)see also Example 5, Method B) (3.30 g, 20.0 mmol), commercial tert-butyldiethyl phosphonoacetate (5.87 mL, 6.31 g, 25.0 mmol), and anhydrouslithium bromide (LiBr) (2.61 g, 30.0 mmol) in a mixture of triethylamine(Et₃N, TEA) (3.83 mL, 2.78 g, 27.5 mmol) and acetonitrile (MeCN) (30mL). Aqueous work-up and purification by silica gel columnchromatography with mixtures of ethyl acetate (EtOAc) and hexane affordthe title compound (9a).

Step B: tert-Butyl 3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate (9b)

Following the General Procedure of Description 3, tert-butyl3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate (9b) is prepared fromtert-butyl (E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (9a) (3.95 g,15.0 mmol) in a mixture of nitromethane (MeNO₂) (10 mL, 11.4 g, 187mmol) and acetonitrile (MeCN) (15 mL) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.24 mL, 2.28 g, 15.3 mmol).Aqueous work-up and purification by silica gel column chromatographywith mixtures of ethyl acetate (EtOAc) and hexane afford the titlecompound (9b).

Step C: tert-Butyl 4-amino-3-(5-amino-2-methyl-phenyl)butanoate (9c)

Following the General Procedure of Description 16, tert-butyl4-amino-3-(5-amino-2-methyl-phenyl)butanoate (9c) is prepared by globalreduction of tert-butyl 3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate(9b) (3.24 g, 10.0 mmol) in the presence of 10 wt-% palladium oncharcoal (Pd/C) containing ˜50 wt-% water (˜1.5 g) in methanol (MeOH)(40 mL) and under an atmosphere of hydrogen (˜15 psi, H₂-balloon). Thecrude material, after filtration over Celite®545, may be used directlyand without further isolation in the next step or may be purified bypreparative HPLC to afford the title compound (9c).

Step D: tert-Butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)butanoate (9d)

Following the General Procedure of Description 15, tert-butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)butanoate (9d)is prepared from tert-butyl 4-amino-3-(5-amino-2-methyl-phenyl)butanoate(9c) (2.64 g, 10.0 mmol) in anhydrous dichloromethane (DCM) (30 mL) atabout 0° C. (ice bath) with di-tert-butyl dicarbonate (Boc₂O) (2.29 g,10.5 mmol) in the presence of triethylamine (Et₃N, TEA) (1.67 mL, 1.21g, 12.0 mmol) and a catalytic amount of 4-(N,N-dimethylamino)pyridine(DMAP) (61 mg, 0.5 mmol). Aqueous work-up and purification by silica gelcolumn chromatography with mixtures of ethyl acetate (EtOAc) and hexaneafford the title compound (9d).

Step E: tert-Butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9e)

Variant A:

Following General Procedure of Description 18, tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9e) is prepared from tert-butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)butanoate (9d)(3.65 g, 10.0 mmol) through reaction with ethylene oxide (12.5 mL, 11.0g, 100.0 mmol) in 15 mL of 50 vol.-% aqueous acetic acid (HOAc) for 24hours at room temperature to yield the title compound (9e) after aqueouswork-up and purification by silica gel chromatography.

Variant B:

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Coggiola, et al., Bioorg. Med. Chem. Lett., 2005,15(15), 3551-3554; Verny et al., Cmpds Radiopharm., 1988, 25(9),949-955; and Lin, Bioorg. Med. Chem. Lett., 2011, 21(3), 940-943), areaction mixture of tert-butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)butanoate (9d)(3.65 g, 10.0 mmol) and commercial 2-chloroethanol (2.68 mL, 3.22 g,40.0 mmol) or commercial 2-bromoethanol (2.84 mL, 5.0 g, 40.0 mmol),calcium carbonate (CaCO₃) (2.0 g, 20.0 mmol, 2.0 equivalents) in water(about 35 mL), and a catalytic amount of potassium iodide (KI) (166 mg,1.0 mmol, 10 mol-%) is heated at reflux for about 12-24 hours. Thereaction is followed by TLC and/or LC/MS to completion. Additionalamounts of 2-halogeno ethanol and CaCO₃ may be added and the reactiontimes may be extended an additional 8-24 h for completion. The pH of thereaction mixture is adjusted to ˜7 with a 2.5 M (10 wt-%) aqueoussolution of sodium hydroxide (NaOH). The aqueous phase is extracted withethyl acetate (4×) and the combined organic extracts are washed with 1.0M hydrochloric acid, a saturated aqueous solution of sodium hydrogencarbonate (NaHCO₃), and brine, dried over anhydrous magnesium sulfate(MgSO₄), filtered, and the solvents removed under reduced pressure usinga rotary evaporator to yield the target compound tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9e). The crude residue may be further purified by silica gel columnchromatography using EtOAc, methanol (MeOH), dichloromethane (DCM), andhexanes, or mixtures of any of the foregoing to furnish the purifiedtarget compound.

Step F: tert-Butyl3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9f)

Following the General Procedure of Description 20 (Variant A),tert-butyl3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9f) is prepared from tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9e) (2.26 g, 5.0 mmol) and methanesulfonyl anhydride (Ms₂O) (3.48 g,20.0 mmol) in the presence of triethylamine (TEA, Et₃N) (3.48 mL, 2.54g, 25.0 mmol) and 4-N,N-(dimethylamino)pyridine (DMAP) (122 mg, 1.0mmol, 20 mol-%) in anhydrous dichloromethane (DCM) (30 mL) to yield thetarget compound (9f) after aqueous work-up and purification by silicagel column chromatography.

Variant B:

Following the General Procedure of Description 20 (Variant B),tert-butyl3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9f) is prepared from tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9e) (2.26 g, 5.0 mmol) and methanesulfonyl chloride (MsCl) (0.96 mL,1.44 g, 12.5 mmol) in the presence of triethylamine (TEA, Et₃N) (2.10mL, 1.52 g, 15.0 mmol) or pyridine (4.0 mL, 3.96 g, 50.0 mmol) inanhydrous dichloromethane (DCM) (30 mL) to yield the target compound(9f) after aqueous work-up and purification by silica gel columnchromatography.

Step G:4-Amino-3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (9)

Following the General Procedure of Description 23 (Variant B),4-amino-3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (9) is prepared from tert-butyl3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-butanoate(9f) (453 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2 N HCl in Et₂O)(10 mL, 20 mmol) to yield the target compound (9) as a soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 104-Amino-3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoic acid (10)

Step A: tert-Butyl3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(10a)

Following the General Procedure of Description 21, tert-butyl3-[5-[bis(2-bromoethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(10a) is prepared from (tert-butyl 3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9f) (1.22 g, 2.0 mmol) through reaction with lithium bromide (LiBr)(1.74 g, 20.0 mmol) in tetrahydrofuran (THF) (10 mL) at refluxtemperature for about 6 hours to yield the title compound (10a) afteraqueous work-up and purification by silica gel column chromatography.

Step B: 4-Amino-3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoicacid (10)

Following the General Procedure of Description 23 (Variant A)4-amino-3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoic acid (10)is prepared from tert-butyl3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(10a) (578 mg, 1.0 mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA/DCM=1:1 v/v, 10 mL) at roomtemperature for about 6 hours to yield the target compound (10) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution.

Following the General Procedure of Description 22,4-amino-3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoic acid (10)is prepared from tert-butyl3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(10a) (578 mg, 1.0 mmol) by hydrolysis in 3.0 M hydrobromic acid (HBr)(about 10 mL) at reflux temperature for about 20 hours to afford thetarget compound (10) as a dihydrobromide salt after evaporation andlyophilization from an aqueous acetonitrile solution. The material maybe further purified by preparative RP-HPLC using awater/acetonitrile/0.1 vol-% formic acid gradient followed bylyophilization in the presence of 1.0 equivalent or an excess of 1.0 Mhydrobromic acid (HBr).

Example 11 4-Amino-3-[5-(bis(2-iodoethyl)amino)-2-methyl-phenyl]butanoicacid (11)

Step A: tert-Butyl 3-[5-(bis(2-iodoethyl)amino)-2-methyl-phenyl]-4-(tertbutoxycarbonylamino)butanoate (11a)

Following the General Procedure of Description 21, tert-butyl3-[5-[bis(2-iodoethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(11a) is prepared from (tert-butyl 3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9f) (1.22 g, 2.0 mmol) through reaction with sodium iodide (NaI) (3.00g, 20.0 mmol) in acetone (10 mL) at reflux temperature for about 6 hoursto yield the title compound (11a) after work-up and purification bysilica gel column chromatography.

Step B: 4-Amino-3-[5-(bis(2-iodoethyl)amino)-2-methyl-phenyl]butanoicacid (11)

Following the General Procedure of Description 22,4-amino-3-[5-(bis(2-iodoethyl)amino)-2-methyl-phenyl]butanoic acid (11)is prepared from tert-butyl3-[5-(bis(2-iodoethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(11a) (672 mg, 1.0 mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA:DCM=1:1, 10 mL) at roomtemperature for about 6 hours to yield the target compound (11) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution. The material may be further purified bypreparative RP-HPLC followed using a water/acetonitrile/0.1 vol-% formicacid gradient followed by lyophilization in the presence of 1.0equivalent or an excess of 1.0 M hydroiodic acid (HI) under exclusion oflight.

Example 124-Amino-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoic acid (12)

Step A: tert-Butyl4-(tert-butoxycarbonylamino)-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate(12a)

Following the General Procedure of Description 21, tert-butyl4-(tert-butoxycarbonylamino)-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate(12a) is prepared from tert-butyl 3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9f) (1.22 g, 2.0 mmol) through reaction with lithium chloride (LiCl)(93 mg, 2.2 mmol) in anhydrous acetonitrile (MeCN) (10 mL) at refluxtemperature for 1.5 hours to yield the title compound (12a) afteraqueous work-up and purification by silica gel column chromatography.

Step B:4-Amino-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (12)

Following the General Procedure of Description 23 (Variant A),4-amino-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (12) is prepared from tert-butyl4-(tert-butoxycarbonylamino)-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate(12a) (550 mg, 1.0 mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA/DCM=1:1 v/v, 10 mL) at roomtemperature for about 6 hours to yield the target compound (12) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution.

Following the General Procedure of Description 23 (Variant B),4-amino-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (12) is prepared from tert-butyl4-(tert-butoxycarbonylamino)-3-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate(12a) (550 mg, 1.00 mmol) in 2 N HCl in diethyl ether (2 N HCl in Et₂O)(10 mL, 20 mmol) to yield the target compound (12) as a soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous acetonitrile solution. The material maybe further purified by preparative RP-HPLC followed using awater/acetonitrile/0.1 vol-% formic acid gradient followed bylyophilization in the presence of 1.0 equivalent or an excess of 1.0 Mhydrochloric acid (HCl).

Example 134-Amino-3-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]butanoicacid (13)

Step A: tert-Butyl3-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(13a)

Following the General Procedure of Description 21, tert-butyl3-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(13a) is prepared from tert-butyl3-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(9f) (1.10 g, 2.0 mmol) through reaction with lithium chloride (LiBr)(191 mg, 2.2 mmol) in anhydrous acetonitrile (MeCN) (10 mL) at refluxtemperature for about 1.5 hours to yield the title compound (13a) afteraqueous work-up and purification by silica gel column chromatography.

Step B:4-Amino-3-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]butanoicacid (13)

Following the General Procedure of Description 23 (Variant A),4-amino-3-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]butanoicacid (13) is prepared from tert-butyl3-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(13a) (534 mg, 1.0 mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA/DCM=1:1 v/v, 10 mL) at roomtemperature for about 6 hours to yield the target compound (13) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution. The material may be further purified bypreparative RP-HPLC followed using a water/acetonitrile/0.1 vol-% formicacid gradient followed by lyophilization.

Example 144-Amino-3-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoic acid (14)

Step A: tert-Butyl3-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(14a)

Adapting literature known protocols (Emmons and Ferris, J. Am Chem. Soc.1953, 75(9), 2257-2257), tert-butyl3-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(14a) is prepared from tert-butyl3-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(10a) (1.16 g, 2.0 mmol) with silver methanesulfonate (silver mesylate,AgOMs) (365 mg, 1.8 mmol) in anhydrous acetonitrile (MeCN) (8 mL) atreflux temperature for about 1 hour under exclusion of light. Aqueouswork-up and purification by silica gel column chromatography withmixtures of ethyl acetate (EtOAc) and hexane afford the title compound(14a).

Step B:4-Amino-3-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methylphenyl]butanoic acid (14)

Following the General Procedure of Description 23 (Variant A),4-amino-3-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (14) is prepared from tert-butyl4-(tert-butoxycarbonylamino)-3-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate (14a) (544 mg, 1.0mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA/DCM=1:1 v/v, 10 mL) at roomtemperature for about 6 hours to yield the target compound (14) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution. The material may be further purified bypreparative RP-HPLC followed using a water/acetonitrile/0.1 vol-% formicacid gradient followed by lyophilization.

Example 154-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid(15)

Step A: Methyl (E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (15a)

Methyl (E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (15a) is preparedfrom commercial 2-methyl-5-nitro-benzaldehyde (5j) (for a synthesis of(5j) see also Example 5, Method B) and commercial trimethylphosphonoacetate as described in Example 2.

Step B: Methyl 3-(2-methyl-5-nitro-phenyl)-4-nitro-pentanoate (15b)

Following the General Procedure of Description 3, methyl3-(2-methyl-5-nitro-phenyl)-4-nitro-pentanoate (15b) is prepared frommethyl (E)-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (15a) (3.38 g, 15.3mmol) in a mixture of nitroethane (EtNO₂) (10.7 mL, 11.3 g, 150 mmol)and acetonitrile (MeCN) (15 mL) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.24 mL, 2.28 g, 15.0 mmol).Aqueous work-up and purification by silica gel column chromatographywith mixtures of ethyl acetate (EtOAc) and hexane afford the titlecompound (15b) as a mixture of diastereomers which may be separated.

Step C: 4-(5-Amino-2-methyl-phenyl)-5-methyl-pyrrolidin-2-one (15c)

Following the General Procedure of Description 4,4-(5-amino-2-methyl-phenyl)-5-methyl-pyrrolidin-2-one (15c) is preparedfrom methyl 3-(2-methyl-5-nitro-phenyl)-4-nitro-pentanoate (15b) (2.96g, 10.0 mmol), freshly washed active Raney®-3202 nickel (about 10 mL ofslurry) in ethanol (EtOH) (about 85 mL) using a Parr hydrogenationapparatus under about 50 psi hydrogen pressure. After filtration overCelite® 545 and completion of the lactamization through heating intoluene at about 95° C. (oil bath temperature), purification by silicagel column chromatography with a mixture of dichloromethane (DCM) andmethanol (MeOH) affords the title compound (15c) as a mixture ofdiastereomers which may be separated.

Step D:4-[5-[Bis(2-chloroethyl)amino]-2-methyl-phenyl]-5-methyl-pyrrolidin-2-one(15d)

Following the General Procedure of Description 17 (Variant A),4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-5-methyl-pyrrolidin-2-one(15d) is prepared from4-(5-amino-2-methyl-phenyl)-5-methyl-pyrrolidin-2-one (15c) (408 mg, 2.0mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (1.27 mL, 10.0mmol), and sodium cyanoborohydride (NaBH₃CN) (503 mg, 8.0 mmol) in amixture of methanol (MeOH) (8 mL) and trifluoroacetic acid (TFA) (4 mL).Aqueous work-up and purification by silica gel column chromatographywith ethyl acetate (EtOAc) and hexane mixtures afford the title compound(15d) as a mixture of diastereomers which may be separated.

Step E: 4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]pentanoicacid (15)

Following the General Procedure of Description 22,4-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]pentanoic acid(15) is prepared from4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-5-methyl-pyrrolidin-2-one(15d) (329 mg, 1.0 mmol) by hydrolysis in a mixture of concentratedhydrochloric acid (HCl) (about 6 mL) and 1.4-dioxane (about 6 mL) atreflux temperature for about 14 hours to afford the title compound (15)as a dihydrochloride salt after isolation using evaporation andlyophilization. The material obtained is purified by preparative RP-HPLCusing a water/acetonitrile/0.1 vol-% formic acid gradient to afford thetitle compound (15) as a dihydrochloride salt after final lyophilizationof the solvents in the presence of an excess of 1.0 M hydrochloric acid(HCl). The diastereomers may be separated simultaneously during thepurification step.

Example 164-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-2-fluoro-butanoicacid (16)

Step A: tert-Butyl-2-fluoro-3-(2-methyl-5-nitro-phenyl)prop-2-enoate(16a)

Following the General Procedure of Description 2 (Variant A),tert-butyl-2-fluoro-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (16a) isprepared from commercial 2-methyl-5-nitro-benzaldehyde (5j) (for asynthesis of (5j) see also Example 5, Method B) (3.30 g, 20.0 mmol),commercial tert-Butyl 2-diethoxyphosphoryl-2-fluoro-acetate (3.71 g,25.0 mmol), and anhydrous lithium bromide (LiBr) (2.61 g, 30.0 mmol) ina mixture of triethylamine (Et₃N, TEA) (3.83 mL, 2.78 g, 27.5 mmol) andacetonitrile (MeCN) (30 mL). Aqueous work-up and purification by silicagel column chromatography with mixtures of ethyl acetate (EtOAc) andhexane afford the title compound (16a). The mixture of (E)/(Z)-isomersmay be separated.

Step B: tert-Butyl2-fluoro-3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate (16b)

Following the General Procedure of Description 3, tert-butyl2-fluoro-3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate (16b) is preparedfrom tert-butyl-2-fluoro-3-(2-methyl-5-nitro-phenyl)prop-2-enoate (16a)(4.22 g, 15.0 mmol) in a mixture of nitromethane (MeNO₂) (10 mL, 11.4 g,187 mmol) and acetonitrile (MeCN) (15 mL) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.24 mL, 2.28 g, 15.0 mmol).Aqueous work-up and purification by silica gel column chromatographywith mixtures of ethyl acetate (EtOAc) and hexane afford the titlecompound (16b) as a mixture of diastereomers, which may be separated.

Step C: tert-Butyl4-amino-3-(5-amino-2-methyl-phenyl)-2-fluoro-butanoate (16c)

Following the General Procedure of Description 16, tert-butyl4-amino-3-(5-amino-2-methyl-phenyl)-2-fluoro-butanoate (16c) is preparedby global reduction of tert-butyl2-fluoro-3-(2-methyl-5-nitro-phenyl)-4-nitro-butanoate (16b) (3.42 g,10.0 mmol) in the presence of 10 wt-% palladium on charcoal (Pd/C)containing ˜50 wt-% water (˜1.5 g) in methanol (MeOH) (40 mL) and underan atmosphere of hydrogen (˜15 psi, H₂-balloon). The crude materialafter filtration may be used directly and without further isolation inthe next step or may be purified by preparative HPLC to afford the titlecompound (16c). The mixture of diastereomers may be separated.

Step D: tert-Butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-2-fluoro-butanoate(16d)

Following the General Procedure of Description 15, tert-butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-2-fluoro-butanoate(16d) is prepared from tert-butyl4-amino-3-(5-amino-2-methyl-phenyl)-2-fluoro-butanoate (2.82 g, 10.0mmol) in anhydrous dichloromethane (DCM) (30 mL) at about 0° C. (icebath) with di-tert-butyl dicarbonate (Boc₂O) (2.29 g, 10.5 mmol) in thepresence of triethylamine (Et₃N, TEA) (1.67 mL, 1.21 g, 12.0 mmol) and acatalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP) (61 mg, 0.5mmol). Aqueous work-up and purification by silica gel columnchromatography with mixtures of ethyl acetate (EtOAc) and hexane affordthe title compound (16d) as a mixture of diastereomers which may beseparated.

Step E: tert-Butyl3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-2-fluoro-butanoate(16e)

Following the General Procedure of Description 17 (Variant A),tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-2-fluoro-butanoate(16e) is prepared from tert-butyl3-(5-amino-2-methyl-phenyl)-4-(tert-butoxycarbonylamino)-2-fluoro-butanoate(16d) (765 mg, 2.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (1.27 mL, 10.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (503 mg,8.0 mmol) in a mixture of methanol (MeOH) (8 mL) and trifluoroaceticacid (TFA) (4 mL). Aqueous work-up and purification by silica gel columnchromatography with ethyl acetate (EtOAc) and hexane mixtures afford thetitle compound (16e) as a mixture of diastereomers, which may beseparated.

Step F:4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-2-fluoro-butanoicacid (16)

Following the General Procedure of Description 22,4-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-2-fluoro-butanoicacid (16) is prepared from tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-2-fluoro-butanoate(16e) (508 mg, 1.0 mmol) by hydrolysis in a mixture of concentratedhydrochloric acid (HCl) (about 6 mL) and 1.4-dioxane (about 6 mL) atreflux temperature for about 14 hours to afford the title compound (16)as a dihydrochloride salt after isolation using evaporation andlyophilization. The material thus obtained is purified by preparativeRP-HPLC using a water/acetonitrile/0.1 vol-% formic acid gradient toafford the title compound (16) as a dihydrochloride salt after finallyophilization of the solvents in the presence of an excess of 1.0 Mhydrochloric acid (HCl). The diastereomers may be separatedsimultaneously during the purification step.

Example 173-(Aminomethyl)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-hydroxy-butanoicacid (17)

Step A:2-(tert-Butyl(dimethyl)silyl)oxy-2-(2-methyl-5-nitro-phenyl)acetaldehyde(17a)

Adapting literature known protocols (International ApplicationPublication No. WO 2002/066410; and Effenberger et al., Chem. Ber.,1991, 124(7), 1651-1659),2-(tert-Butyl(dimethyl)silyl)oxy-2-(2-methyl-5-nitro-phenyl)acetaldehyde(17a) is prepared from commercial 2-methyl-5-nitro-benzaldehyde (5j)(for a synthesis of (5j) see also Example 5, Method B) following asequence of i) cyanohydrin reaction, ii) hydrolysis, iii)esterification, iv) O-silylether protection, and v) ester reduction.

Step B: tert-Butyl(E)-4-(tert-butyl(dimethyl)silyl)oxy-4-(2-methyl-5-nitro-phenyl)but-2-enoate(17b)

Following the General Procedure of Description 2 (Variant A), tert-butyl(E)-4-(tert-butyl(dimethyl)silyl)oxy-4-(2-methyl-5-nitro-phenyl)but-2-enoate(17b) is prepared from2-(tert-Butyl(dimethyl)silyl)oxy-2-(2-methyl-5-nitro-phenyl)acetaldehyde(17a) (6.19 g, 20.0 mmol), commercial tert-butyl diethylphosphonoacetate (5.87 mL, 6.31 g, 25.0 mmol), and anhydrous lithiumbromide (LiBr) (2.61 g, 30.0 mmol) in a mixture of triethylamine (Et₃N,TEA) (3.83 mL, 2.78 g, 27.5 mmol) and acetonitrile (MeCN) (30 mL).Aqueous work-up and purification by silica gel column chromatographywith mixtures of ethyl acetate (EtOAc) and hexane afford the titlecompound (17b) as a mixture of geometric (E)/(Z)-isomers, which may beseparated.

Step C: tert-Butyl4-(tert-butyl(dimethyl)silyl)oxy-4-(2-methyl-5-nitro-phenyl)-3-(nitro-methyl)butanoate(17c)

Following the General Procedure of Description 3, tert-butyl4-(tert-butyl(dimethyl)silyl)oxy-4-(2-methyl-5-nitro-phenyl)-3-(nitromethyl)butanoate(17c) is prepared from tert-butyl(E)-4-(tert-butyl(dimethyl)silyl)oxy-4-(2-methyl-5-nitro-phenyl)but-2-enoate(17b) (6.11 g, 15.0 mmol) in a mixture of nitromethane (MeNO₂) (10 mL,11.4 g, 187 mmol) and acetonitrile (MeCN) (15 mL) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.24 mL, 2.28 g, 15.3 mmol).Aqueous work-up and purification by silica gel column chromatographywith mixtures of ethyl acetate (EtOAc) and hexane afford the titlecompound (17c) as a mixture of diastereomers, which may be separated.

Step D: tert-Butyl3-(aminomethyl)-4-(5-amino-2-methyl-phenyl)-4-(tert-butyl(dimethyl)-silyl)oxy-butanoate(17d)

Adapting literature known protocols (Osby and Ganem, Tetrahedron Lett.,1985, 26(52), 6413-6416), a mixture of nickel(II) dichloride hexahydrate(NiCl₂.6H₂O) (1.19 g, 5.0 mmol) in methanol (MeOH) (80 mL) is sonicatedto effect complete dissolution. Solid sodium borohydride (NaBH₄) (567mg, 15.0 mmol) is added in small portions upon a black precipitate(Ni₂B) is generated immediately and hydrogen gas is generated(exotherm!). After about 30 min, a solution of tert-butyl4-(tert-butyl(dimethyl)silyl)oxy-4-(2-methyl-5-nitro-phenyl)-3-(nitromethyl)butanoate(17c) (4.69 g, 10.0 mmol) in MeOH (20 mL) is added followed byportion-wise addition of more NaBH₄ (1.13 g, 30.0 mmol) at roomtemperature. The reaction is monitored by TLC and/or LCMS tillcompletion. The reaction mixture is filtered over Celite® 545 and thefilter residue is washed with additional MeOH. The combined filtratesare evaporated to dryness under reduced pressure using a rotaryevaporator. The crude tert-butyl3-(aminomethyl)-4-(5-amino-2-methyl-phenyl)-4-(tert-butyl(dimethyl)silyl)oxy-butanoate(17d) is used directly in the next step without further isolation andpurification procedures.

Step E: tert-Butyl4-(5-amino-2-methyl-phenyl)-3-[(tert-butoxycarbonylamino)methyl]-4-(tert-butyl(dimethyl)silyl)oxy-butanoate(17e)

Following the General Procedure of Description 15, tert-butyl4-(5-amino-2-methyl-phenyl)-3-[(tert-butoxycarbonylamino)methyl]-4-(tert-butyl(dimethyl)silyl)oxy-butanoate(17e) is prepared from tert-butyl3-(aminomethyl)-4-(5-amino-2-methyl-phenyl)-4-(tert-butyl(dimethyl)silyl)oxy-butanoate(17d) (4.09 g, 10.0 mmol) in anhydrous dichloromethane (DCM) (30 mL) atabout 0° C. (ice bath) with di-tert-butyl dicarbonate (Boc₂O) (2.29 g,10.5 mmol) in the presence of triethylamine (Et₃N, TEA) (1.67 mL, 1.21g, 12.0 mmol) and a catalytic amount of 4-(N,N-dimethylamino)pyridine(DMAP) (61 mg, 0.5 mmol). Aqueous work-up and purification by silica gelcolumn chromatography with mixtures of ethyl acetate (EtOAc) and hexaneafford the title compound (17e) as a mixture of diastereomers, which maybe separated.

Step F: tert-Butyl4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-[(tert-butoxycarbonylamino)methyl]-4-(tert-butyl(dimethyl)silyl)oxy-butanoate(17f)

Following the General Procedure of Description 17 (Variant B),tert-butyl4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-[(tert-butoxycarbonylamino)methyl]-4-(tert-butyl(dimethyl)silyl)oxy-butanoate(17f) is prepared from tert-butyl4-(5-amino-2-methyl-phenyl)-3-[(tert-butoxycarbonylamino)methyl]-4-(tert-butyl(dimethyl)silyl)oxy-butanoate(17e) (1.02 g, 2.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (1.27 mL, 10.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (503 mg,8.0 mmol) in a mixture of methanol (MeOH) (6 mL) and acetic acid (HOAc)(6 mL). Aqueous work-up and purification by silica gel columnchromatography with ethyl acetate (EtOAc) and hexane mixtures afford thetitle compound (17f) as a mixture of diastereomers, which may beseparated.

Step G:3-(Aminomethyl)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-hydroxy-butanoicacid (17)

Following the General Procedure of Description 22,3-(aminomethyl)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-hydroxy-butanoicacid (17) is prepared from tert-butyl4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-[(tert-butoxycarbonylamino)methyl]-4-(tert-butyl(dimethyl)silyl)oxy-butanoate(17f) (634 mg, 1.0 mmol) by hydrolysis in a mixture of concentratedhydrochloric acid (HCl) (about 6 mL) and 1.4-dioxane (about 6 mL) atabout 60° C. (oil bath temperature) for about 8 hours to afford thetitle compound (17) as a dihydrochloride salt after isolation usingevaporation and lyophilization. The material obtained is purified bypreparative RP-HPLC using a water/acetonitrile/0.1 vol-% formic acidgradient to afford the title compound (17) as a dihydrochloride saltafter final lyophilization of the solvents in the presence of an excessof 1.0 M hydrochloric acid (HCl). The diastereomers may be separatedsimultaneously during the purification step.

Example 184-Amino-3-[5-[bis(2-chloroethyl)amino]-2-nitro-phenyl]butanoic acid (18)

Step A: Methyl (E)-3-(5-chloro-2-nitro-phenyl)prop-2-enoate (18a)

Following the General Procedure of Description 2 (Variant A), methyl(E)-3-(5-chloro-2-nitro-phenyl)prop-2-enoate (18a) is prepared fromcommercial 5-chloro-2-nitro-benzaldehyde (6.50 g, 35.0 mmol), trimethylphosphonoacetate (7.08 mL, 7.97 g, 43.75 mmol), and anhydrous lithiumbromide (LiBr) (4.56 g, 52.5 mmol) in a mixture of triethylamine (Et₃N,TEA) (6.71 mL, 4.87 g, 48.1 mmol) and acetonitrile (MeCN) (35 mL).Aqueous work-up and purification by silica gel column chromatographyusing ethyl acetate (EtOAc) and hexane mixtures afford the titlecompound (18a).

Step B: 3-(5-Chloro-2-nitro-phenyl)pentanedioic acid (18b) Step i:Trimethyl 2-(5-chloro-2-nitro-phenyl)propane-1,1,3-tricarboxylate (18b′)

Adapting literature known protocols (Liu, et al., Tetrahedron:Asymmetry, 2001, 12, 419-426 and Stowe, et al., Org. Lett., 2010, 12(4),756-759), a solution of sodium methoxide (MeONa) is prepared bydissolving sodium metal (Na) (300 mg, 13.0 mol) in anhydrous methanol(MeOH) (10 mL) at about 0° C. (ice bath) under a nitrogen atmosphere. Asolution of dimethyl malonate (1.49 mL, 1.72 g, 13.0 mol) is addedfollowed by a solution of methyl(E)-3-(5-chloro-2-nitro-phenyl)prop-2-enoate (18a) (2.42 g, 10.0 mol) inMeOH (10 mL) after about 30 min. The reaction mixture is graduallywarmed to room temperature and subsequently heated at reflux (75° C. oilbath temperature). The reaction is monitored by TLC and/or analyticalLC/MS tocompletion. The reaction is evaporated to dryness under reducedpressure using a rotary evaporator. Aqueous work-up and purification bysilica gel column chromatography using ethyl acetate (EtOAc) and hexanesmixtures afford trimethyl2-(5-chloro-2-nitrophenyl)propane-1,1,3-tricarboxylate (18b′).

Step ii: 3-(5-Chloro-2-nitro-phenyl)pentanedioic acid (18b)

Variant A:

Adapting literature known protocols (Liu, et al., Tetrahedron:Asymmetry, 2001, 12, 419-426), 3-(5-chloro-2-nitro-phenyl)pentanedioicacid (18b) is prepared by heating a suspension of trimethyl2-(5-chloro-2-nitro-phenyl)propane-1,1,3-tricarboxylate (18b′) (3.74 g,10.0 mol) in 1.0 M aqueous sodium hydroxide (NaOH) (10 mL) to gentlereflux for about 12 h (105° C. oil bath temperature) followed byacidifying with concentrated hydrochloric acid (HCl) to pH 0-1 at aboutroom temperature. The solution is then heated to reflux for about 12 h(105° C. oil bath temperature) to effect complete decarboxylation. Thereaction is monitored by TLC and/or analytical LC/MS to completion. Theaqueous solution is distilled to remove most of the water and extractedwith ethyl acetate. Aqueous work-up and purification by crystallizationfrom ethyl acetate (EtOAc) and hexanes mixtures afford the titlecompound (18b).

Variant B:

Adapting literature known protocols (Krapcho, Synthesis, 1982, 805-822 &893-914; and Stowe, et al., Org. Lett., 2010, 12(4), 756-759), asolution of trimethyl2-(5-chloro-2-nitro-phenyl)propane-1,1,3-tricarboxylate (18b′) (3.74 g,10.0 mol) and sodium chloride (NaCl) (292 mg, 5.0 mmol) in a mixture ofdimethylsulfoxide (DMSO) (23 mL) and water (780 μL) is heated to refluxfor about 12 h. The reaction is monitored by TLC and/or analytical LC/MSto completion. Aqueous work-up and purification by silica gel columnchromatography using ethyl acetate (EtOAc) and hexanes mixtures afforddimethyl 3-(5-chloro-2-nitro-phenyl)pentanedioate (18b″). Adaptingliterature known protocols (Nejman, et al., Tetrahedron, 2005, 61,8536-854), 3-(5-chloro-2-nitro-phenyl)pentanedioic acid (18b) isprepared heating a solution of dimethyl3-(5-chloro-2-nitro-phenyl)pentanedioate (18b″) (3.16 g, 10.0 mmol) in amixture of 6.0 M hydrochloric acid (HCl) and 1,4-dioxane for about 12 hto reflux (105° C. oil bath temperature) to effect complete esterhydrolysis and decarboxylation. The reaction is monitored by TLC and/oranalytical LC/MS to completion. The solvents are distilled off underreduced pressure using a rotary evaporator. Aqueous work-up andpurification by crystallization from ethyl acetate (EtOAc) and hexanesmixtures afford the title compound (18b).

Step C: 4-(5-Chloro-2-nitro-phenyl)tetrahydropyran-2,6-dione (18c)

Adapting literature known protocols (Sulyok, J. Med. Chem., 2001,44(12), 1938-1950; Ji, et al., Tetrahedron Lett., 2009, 50(45),6166-6168; and Liu, et al., Tetrahedron: Asymmetry, 2001, 12, 419-426),4-(5-chloro-2-nitro-phenyl)tetrahydropyran-2,6-dione (18c) is preparedby heating 3-(5-chloro-2-nitro-phenyl)pentanedioic acid (18b) (2.88 g,10.0 mmol) in acetic anhydride (Ac₂O) (2.83 mL, 3.06 g, 30 mmol) atreflux (about 130° C. oil bath temperature). After cooling to roomtemperature, the title compound (18c) is precipitated with diethyl ether(Et₂O) the solid collected by filtration, washed with cold Et₂O anddried under reduced pressure to remove residual solvents.

Step D: 5-Amino-3-(5-chloro-2-nitro-phenyl)-5-oxo-pentanoic acid (18d)

Adapting literature known protocols (Ji, et al., Tetrahedron Lett.,2009, 50(45), 6166-6168; and Hoekstra, et al., Org. Proc. Dev. Dev.,1997, 1(1), 26-38), 5-amino-3-(5-chloro-2-nitro-phenyl)-5-oxo-pentanoicacid (18d) is prepared from4-(5-chloro-2-nitro-phenyl)tetrahydropyran-2,6-dione (18a) (2.70 g, 10.0mmol) in tetrahydrofuran (THF) (20 mL) with a concentrated solution ofammonia (NH₃) in methanol (MeOH) (˜7.0 M; about 100 mL) at roomtemperature for about 14 hours. The reaction course is followed by TLCand/or LC/MS till completion. The reaction mixture is concentrated todryness under reduced pressure using a rotary evaporator. The residue istriturated with ethyl acetate (EtOAc) (2×). The solids remaining aftertitruation are acidified at a temperature of about 0° C. (ice bath) with1.0 M hydrochloric acid. Aqueous work-up yields the title compound(18d). The crude material may either be used directly in the next stepor may be further purified by silica gel column chromatography usingmethanol (MeOH), dichloromethane (DCM), and hexane mixtures or byre-crystallization.

Step E: Methyl 5-amino-3-(5-chloro-2-nitro-phenyl)-5-oxo-pentanoate(18e)

Adapting literature known protocols, methyl5-amino-3-(5-chloro-2-nitro-phenyl)-5-oxo-pentanoate (18e) is preparedfrom 5-amino-3-(5-chloro-2-nitro-phenyl)-5-oxo-pentanoic acid (18d)(2.87 g, 10.0 mmol), and iodomethane (MeI) (1.87 mL, 4.26 g, 30.0 mmol)in the presence of potassium carbonate (K₂CO₃) (5.53 g, 40.0 mmol) inanhydrous N,N-dimethylformamide (DMF) (about 30 mL) at room temperature.The reaction is monitored by TLC and/or analytical LC/MS to completion.Aqueous work-up and purification by silica gel column chromatographyusing ethyl acetate (EtOAc) and hexanes mixtures afford the titlecompound (18e).

Step F: 4-(5-Chloro-2-nitro-phenyl)pyrrolidin-2-one (18f)

Adapting literature known protocols (Loudon, et al., J. Org. Chem.,1984, 49(22), 4272-4276; Loudon and Boutin, J. Org. Chem., 1984, 49(22),4277-4286; Nicolaou, et al., Bioorg. Med. Chem. 1998, 6(8), 1185-1208;and Ji et al., Tetrahedron Lett., 2009, 50(45), 6166-6168),4-(5-chloro-2-nitro-phenyl)pyrrolidin-2-one (18f) is prepared frommethyl 5-amino-3-(5-chloro-2-nitro-phenyl)-5-oxo-pentanoate (18e) (2.87g, 10.0 mmol) in a mixture of acetonitrile (MeCN) (15 mL) and water (15mL) through an acidic Hoffmann-rearrangement with[I,I-bis(trifluoroacetoxy)iodo]benzene (phenyl iodosylbis(trifluoroacetate, PIFA) (4.25 g, 10.0 mmol) followed by partialspontaneous intramolecular lactamization of the intermittent amine atroom temperature. The reaction may be monitored by TLC and/or analyticalLC/MS to completion. The reaction mixture is diluted with 2.0 Mhydrochloric acid (HCl). The aqueous phase is extracted with diethylether (Et₂O) (3×) to remove ether-soluble iodobenzene dichloride whichis discarded. The aqueous phase is frozen and the solvents lyophilizedoff to yield an intermediate hydrochloride. The hydrochloride is dilutedwith water and the aqueous phase is adjusted to pH 8-9 with 2.0 Maqueous sodium hydroxide (NaOH). The aqueous phase is extracted withethyl acetate (EtOAc) (3×). The combined organic extracts are eitherpartially evaporated under reduced pressure followed by heating directlyat reflux for about 6 hours to yield the crude title compound (18f).Alternatively, the solvent is evaporated under reduced pressure using arotary evaporator, the residue is diluted with toluene or benzene andthe solution is heated at reflux for about 6 hours. The crude material(18f) may either be used directly in the next step or may be furtherpurified by silica gel chromatography using EtOAc, methanol (MeOH),dichloromethane (DCM), and/or hexanes or by re-crystallization.

Step G: 4-[5-(Bis(2-hydroxyethyl)amino)-2-nitro-phenyl]pyrrolidin-2-one(18g)

Adapting literature known protocols (Atwell, et al., J. Med. Chem.,2007, 50(6), 1197-1212; Palmer, et al., J. Med. Chem., 1994, 37,2175-2184; Palmer, et al., J. Med. Chem., 1992, 35(17), 3214-3222;Palmer, et al., J. Med. Chem, 1990, 33(1), 112-121; Davies, et al., J.Med. Chem. 2005, 48(16), 5321-5328; Jordan, et al., Bioorg. Med. Chem.,2002, 10(8), 2625-2633; Dheyongera, et al., Bioorg. Med. Chem., 2005,13(3), 689-698; Lin, et al., Bioorg. Med. Chem. Lett., 2011, 21(3),940-943; and Ferlin, et al., Bioorg. Med. Chem., 2004, 12(4), 771-777),4-[5-(bis(2-hydroxyethyl)amino)-2-nitro-phenyl]pyrrolidin-2-one (18g) isprepared from 4-(5-chloro-2-nitro-phenyl)pyrrolidin-2-one (18f) (2.41 g,10.0 mmol) through nucleophilic aromatic displacement (SNAr) with2-(2-hydroxyethylamino)ethanol (diethanolamine) (1.92 mL, 2.10 g, 20.0mmol) at a temperature of about 80-140° C. for about 4-12 h. Optionally,the reaction is conducted in an organic solvent, e.g., anhydrousdimethylsulfoxide (DMSO) or 1,4-dioxane (about 20 mL), or in thepresence of a catalyst, e.g., copper powder (64 mg, 1.0 mmol, 10 mol-%).The reaction may be monitored by TLC and/or analytical LC/MS tocompletion. Aqueous work-up and purification by silica gel columnchromatography using ethyl acetate (EtOAc), methanol (MeOH),dichloromethane (DCM), and hexanes mixtures afford the title compound(18g).

Step H: 4-[5-(Bis(2-chloroethyl)amino)-2-nitro-phenyl]pyrrolidin-2-one(18h)

Following the General Procedure of Description 19 (Variant B),4-[5-(bis(2-chloroethyl)amino)-2-nitro-phenyl]pyrrolidin-2-one (18h) isprepared from4-[5-(bis(2-hydroxyethyl)amino)-2-nitro-phenyl]pyrrolidin-2-one (18g)(618 mg, 2.0 mmol) through reaction with phosphoryl chloride (POCl₃)(0.93 mL, 1.53 g, 10.0 mmol) in anhydrous benzene (10 mL) for about 5 hat a temperature of about 80° C. to yield the title compound (18h) afterwork-up and purification by silica gel column chromatography using ethylacetate (EtOAc), methanol (MeOH), dichloromethane (DCM), and hexanesmixtures.

Step I: 4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-nitro-phenyl]butanoicacid (18)

Following the General Procedure of Description 22,4-amino-3-[5-[bis(2-chloroethyl)amino]-2-nitro-phenyl]butanoic acid (18)is prepared from4-[5-(bis(2-chloroethyl)amino)-2-nitro-phenyl]pyrrolidin-2-one (18h)(346 mg, 1.0 mmol) by hydrolysis in a mixture of concentratedhydrochloric acid (HCl) (about 5 mL) and 1,4-dioxane (about 5 mL) atabout 90° C. for about 15 hours to afford the title compound (18) as adihydrochloride salt after isolation using evaporation andlyophilization. The material obtained is purified by preparative RP-HPLCusing a water/acetonitrile/0.1 vol-% formic acid gradient to afford thetitle compound (18) as a dihydrochloride salt after final lyophilizationof the solvents in the presence of 1 equivalent or an excess of 1.0 Mhydrochloric acid (HCl).

Example 19[3-Amino-2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinicacid (19)

Step A: 1-Methyl-4-nitro-2-[(E)-2-nitrovinyl]benzene (19a)

Adapting literature known protocols (U.S. Pat. No. 8,344,028; andKabalka and Varma, Org. Prep. Proced. Int., 1987, 19(4-5), 283-328),1-methyl-4-nitro-2-[(E)-2-nitrovinyl]benzene (19a) is prepared byheating a solution of commercial 2-methyl-5-nitro-benzaldehyde (5j)(1.65 g, 10.0 mmol) (for a synthesis of (5j) see also Example 5, MethodB), ammonium acetate (NH₄OAc) (1.31 g, 17.0 mmol) in a mixture ofnitromethane (MeNO₂) (1.61 mL, 1.83 g, 30.0 mmol) and acetic acid (HOAc)(10.0 mL) to reflux for about 3 hours. Aqueous work-up and purificationby silica gel column chromatography using ethyl acetate (EtOAc) andhexanes mixtures afford the title compound (19a).

Step B:2-[1-[(1,1-Diethoxyethyl(ethoxy)phosphoryl)methyl]-2-nitro-ethyl]-1-methyl-4-nitro-benzene(19b)

Adapting literature known protocols (U.S. Pat. No. 8,344,028; andBaylis, Tetrahedron Lett., 1995, 36(51), 9385-9388),2-[1-[(1,1-diethoxyethyl(ethoxy)phosphoryl)-methyl]-2-nitro-ethyl]-1-methyl-4-nitro-benzene(19b) is prepared from 1-methyl-4-nitro-2-[(E)-2-nitrovinyl]benzene(19a) through 1,4-conjugate addition of lithiated commercial1-[1-ethoxy-1-(ethoxy(methyl)phosphoryl)ethoxy]ethane (Baillie, et al.,U.S. Pat. No. 4,339,443 (1982). To a solution of[1-ethoxy-1-(ethoxy(methyl)phosphoryl)ethoxy]ethane (2.65 mL, 2.69 g,12.0 mmol) in anhydrous tetrahydrofuran (THF) (15 mL) and under anitrogen atmosphere and at a temperature of about −78° C. (dryice/acetone bath) is dropwise added a solution of n-butyllithium (nBuLi)in hexane (12.0 mL, 12.0 mmol). After about 30 min, the solution of thelithiated compound is added via a cannula within about 5 minutes to acooled solution (−78° C.; dry ice/acetone bath, nitrogen atmosphere) of(19a) (2.08 g, 10.0 mmol) in anhydrous tetrahydrofuran (THF) (15 mL).The reaction is stirred at this temperature for about 30 minutesfollowed by gradual warming to about 0° C. (ice/water bath). Stirring iscontinued at this temperature for another 30 minutes. Aqueous work-upand purification by silica gel column chromatography using ethyl acetate(EtOAc) and hexanes mixtures afford the title compound (19b).

Step C:3-[1-(Aminomethyl)-2-(1,1-diethoxyethyl(ethoxy)phosphoryl)ethyl]-4-methyl-aniline(19c)

Adapting a literature known protocol (U.S. Pat. No. 8,344,028) andfollowing the General Procedure of Description 3,3-[1-(aminomethyl)-2-(1,1-diethoxyethyl(ethoxy)phosphoryl)ethyl]-4-methyl-aniline(19c) is prepared from2-[1-[(1,1-diethoxyethyl(ethoxy)phosphoryl)methyl]-2-nitro-ethyl]-1-methyl-4-nitro-benzene(19b) (4.32 g, 10.0 mmol)), freshly washed active Raney®-3202 nickel(about 10 mL of slurry) in ethanol (EtOH) (75 mL) using a Parrhydrogenation apparatus under about 50 psi hydrogen pressure. Aqueouswork-up and purification by silica gel column chromatography using ethylacetate (EtOAc) and hexanes mixtures afford the title compound (19c).

Step D: tert-ButylN-[2-(5-amino-2-methyl-phenyl)-3-(1,1-diethoxyethyl(ethoxy)-phosphoryl)-propyl]carbamate(19d)

Following the General Procedure of Description 15, tert-butylN-[2-(5-amino-2-methyl-phenyl)-3-(1,1-diethoxyethyl(ethoxy)phosphoryl)-propyl]carbamate(19d) is prepared from3-[1-(aminomethyl)-2-(1,1-diethoxyethyl(ethoxy)phosphoryl)ethyl]-4-methyl-aniline(19c) (3.72 g, 10.0 mmol) in anhydrous dichloromethane (DCM) (30 mL) atabout 0° C. (ice bath) with di-tert-butyl dicarbonate (Boc₂O) (2.29 g,10.5 mmol) in the presence of triethylamine (Et₃N, TEA) (1.67 mL, 1.21g, 12.0 mmol) and a catalytic amount of 4-(N,N-dimethylamino)pyridine(DMAP) (61 mg, 0.5 mmol). Aqueous work-up and purification by silica gelcolumn chromatography using ethyl acetate (EtOAc) and hexanes mixturesafford the title compound (19d).

Step E: tert-ButylN-[2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(1,1-diethoxyethyl(ethoxy)phosphoryl)propyl]carbamate (19e)

Following the General Procedure of Description 17 (Variant C),tert-butylN-[2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(1,1-diethoxyethyl(ethoxy)phosphoryl)-propyl]carbamate(19e) is prepared from tert-butylN-[2-(5-amino-2-methyl-phenyl)-3-(1,1-diethoxyethyl(ethoxy)phosphoryl)-propyl]carbamate(19d) (945 mg, 2.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (1.52 mL, 12.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (628 mg,10.0 mmol) in a mixture of methanol (MeOH) (10 mL) and 85 wt-%phosphoric acid (H₃PO₄) (5 mL). Aqueous work-up and purification bysilica gel column chromatography using ethyl acetate (EtOAc) and hexanesmixtures afford the title compound (19e).

Step F:[3-Amino-2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinicacid (19)

Following the General Procedure of Description 22,[3-amino-2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinicacid (19) is prepared from protected β-substituted γ-amino acid analogN-mustard (19e) (598 mg, 1.0 mmol) by hydrolysis in a mixture ofconcentrated hydrochloric acid (HCl) (about 5 mL) and 1,4-dioxane (about5 mL) at about 90° C. for about 15 hours to afford the title compound(20) as a dihydrochloride salt after isolation using evaporation andlyophilization. The material thus obtained is purified by preparativeRP-HPLC using a water/acetonitrile/0.1 vol-% formic acid gradient toafford the title compound (19) as a dihydrochloride salt after finallyophilization of the solvents in the presence of 1 equivalent or anexcess of 1.0 M hydrochloric acid (HCl).

Example 203-[1-(Aminomethyl)-2-(1H-tetrazol-5-yl)ethyl]-N,N-bis(2-chloroethyl)-4-methyl-aniline(20)

Step A: 1-Methyl-4-nitro-2-[(E)-2-nitrovinyl]benzene (19a)

Adapting literature known protocols (U.S. Pat. No. 8,344,028; andKabalka and Varma, Org. Prep. Proced. Int., 1987, 19(4-5), 283-328),1-methyl-4-nitro-2-[(E)-2-nitrovinyl]benzene (19a) is prepared fromcommercial 2-methyl-5-nitro-benzaldehyde (5j) (for a synthesis of (5j)see also Example 5, Method B) as described in Example 19, Step A.

Step B:5-[2-(2-Methyl-5-nitro-phenyl)-3-nitro-propyl]-1-trityl-tetrazole (20b)

Adapting literature known protocols (Huff et al., Tetrahedron Lett.,1996, 37(12), 3655-3658; Baylis, Tetrahedron Lett., 1995, 36(51),9385-9388; and Xu, et al., U.S. Pat. No. 8,344,028 (2013)),5-[2-(2-methyl-5-nitro-phenyl)-3-nitro-propyl]-1-trityl-tetrazole (20b)is prepared from 1-methyl-4-nitro-2-[(E)-2-nitrovinyl]benzene (19a)through 1,4-conjugate addition of lithiated 5-methyl-1-trityl-tetrazole(Huff et al., Tetrahedron Lett., 1996, 37(12), 3655-3658).5-Methyl-1-trityl-tetrazole (3.92 g, 12.0 mmol) in anhydroustetrahydrofuran (THF) (15 mL) is lithiated under a nitrogen atmosphereand at a temperature of about −78° C. (dry ice/acetone bath) withn-butyllithium (nBuLi) in hexane (12.0 mL, 12.0 mmol) and added via acannula to a cooled solution (−78° C.; dry ice/acetone bath, nitrogenatmosphere) of (19a) (2.08 g, 10.0 mmol) in anhydrous tetrahydrofuran(THF) (15 mL). Aqueous work-up and purification by silica gel columnchromatography using ethyl acetate (EtOAc) and hexanes mixtures affordthe title compound (20b).

Step C:3-[1-(Aminomethyl)-2-(1-trityltetrazol-5-yl)ethyl]-4-methyl-aniline(20c)

Adapting a literature known protocol (Osby and Ganem, Tetrahedron Lett.,1985, 26(52), 6413-6416), a mixture of nickel(II) dichloride hexahydrate(NiCl₂.6H₂O) (1.19 g, 5.0 mmol) in methanol (MeOH) (80 mL) is sonicatedto effect complete dissolution. Solid sodium borohydride (NaBH₄) (567mg, 15.0 mmol) is added in small portions upon a black precipitate(Ni₂B) is generated immediately and hydrogen gas is generated(exotherm!). After about 30 min, a solution of5-[2-(2-methyl-5-nitro-phenyl)-3-nitro-propyl]-1-trityl-tetrazole (20b)(5.35 g, 10.0 mmol) in MeOH (20 mL) is added followed by portion-wiseaddition of more NaBH₄ (1.13 g, 30.0 mmol) at room temperature. Thereaction is monitored by TLC and/or LCMS to completion. The reactionmixture is filtered over Celite® 545 and the filter residue is washedwith additional MeOH. The combined filtrates are evaporated to drynessunder reduced pressure using a rotary evaporator. The crude material(20c) is used directly in the next step without further isolation andpurification.

Step D: tert-ButylN-[2-(5-amino-2-methyl-phenyl)-3-(1-trityltetrazol-5-yl)propyl]carbamate(20d)

Following the General Procedure of Description 15, tert-butylN-[2-(5-amino-2-methyl-phenyl)-3-(1-trityltetrazol-5-yl)propyl]carbamate(20d) is prepared from3-[1-(aminomethyl)-2-(1-trityltetrazol-5-yl)ethyl]-4-methyl-aniline(20c) (4.75 g, 10.0 mmol) in anhydrous dichloromethane (DCM) (30 mL) atabout 0° C. (ice bath) with di-tert-butyl dicarbonate (Boc₂O) (2.29 g,10.5 mmol) in the presence of triethylamine (Et₃N, TEA) (1.67 mL, 1.21g, 12.0 mmol) and a catalytic amount of 4-(N,N-dimethylamino)pyridine(DMAP) (61 mg, 0.5 mmol). Aqueous work-up and purification by silica gelcolumn chromatography using ethyl acetate (EtOAc) and hexanes mixturesafford the title compound (20d).

Step E: tert-ButylN-[2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(1H-tetrazol-5-yl)propyl]carbamate(20e)

Following the General Procedure of Description 17 (Variant C),tert-butylN-[2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(1H-tetrazol-5-yl)propyl]carbamate(20e) is prepared from tert-butylN-[2-(5-amino-2-methyl-phenyl)-3-(1-trityltetrazol-5-yl)propyl]carbamate(20d) (1.15 g, 2.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (1.52 mL, 12.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (628 mg,10.0 mmol) in a mixture of methanol (MeOH) (10 mL) and 85 wt-%phosphoric acid (H₃PO₄) (5 mL). Aqueous work-up and purification bysilica gel column chromatography using ethyl acetate (EtOAc) and hexanesmixtures afford the title compound (20e).

Step F:3-[1-(Aminomethyl)-2-(1H-tetrazol-5-yl)ethyl]-N,N-bis(2-chloroethyl)-4-methyl-aniline(20)

Following the General Procedure of Description 22,[3-amino-2-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinicacid (20) is prepared from protected β-substituted γ-amino acid analogN-mustard (20e) (457 mg, 1.0 mmol) by hydrolysis in a mixture ofconcentrated hydrochloric acid (HCl) (about 5 mL) and 1,4-dioxane (about5 mL) at about 60° C. for about 15 hours to afford the title compound(20) as a solid dihydrochloride salt after isolation using evaporationand lyophilization. The material obtained is purified by preparativeRP-HPLC using a water/acetonitrile/0.1 vol-% formic acid gradient toafford the title compound (20) as a dihydrochloride salt after finallyophilization of the solvents in the presence of 1 equivalent or anexcess of 1.0 M hydrochloric acid (HCl).

Example 214-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]butanoic acid(21)

Step A: tert-Butyl (E)-3-(2-methoxy-5-nitro-phenyl)prop-2-enoate (21a)

Following the General Procedure of Description 2 (Variant A), tert-butyl(E)-3-(2-methoxy-5-nitro-phenyl)prop-2-enoate (21a) is prepared fromcommercial 2-methoxy-5-nitro-benzaldehyde (3.62 g, 20.0 mmol),commercial tert-butyl diethyl phosphonoacetate (5.87 mL, 6.31 g, 25.0mmol), and anhydrous lithium bromide (LiBr) (2.61 g, 30.0 mmol) in amixture of triethylamine (Et₃N, TEA) (3.83 mL, 2.78 g, 27.5 mmol) andacetonitrile (MeCN) (30 mL). Aqueous work-up and purification by silicagel column chromatography with mixtures of ethyl acetate (EtOAc) andhexane afford the title compound (21a).

Step B: tert-Butyl 3-(2-methoxy-5-nitro-phenyl)-4-nitro-butanoate (21b)

Following the General Procedure of Description 3, tert-butyl3-(2-methoxy-5-nitro-phenyl)-4-nitro-butanoate (21b) is prepared fromtert-butyl (E)-3-(2-methoxy-5-nitro-phenyl)prop-2-enoate (21a) (4.19 g,15.0 mmol) in a mixture of nitromethane (MeNO₂) (10 mL, 11.4 g, 187mmol) and acetonitrile (MeCN) (15 mL) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.24 mL, 2.28 g, 15.3 mmol).Aqueous work-up and purification by silica gel column chromatographywith mixtures of ethyl acetate (EtOAc) and hexane afford the titlecompound (21b).

Step C: tert-Butyl 4-amino-3-(5-amino-2-methoxy-phenyl)butanoate (21c)

Following the General Procedure of Description 16, tert-butyl4-amino-3-(5-amino-2-methoxy-phenyl)butanoate (21c) is prepared byglobal reduction of tert-butyl3-(2-methoxy-5-nitro-phenyl)-4-nitro-butanoate (21b) (3.40 g, 10.0 mmol)in the presence of 10 wt-% palladium on charcoal (Pd/C) containing ˜50wt-% water (˜1.5 g) in methanol (MeOH) (40 mL) and under an atmosphereof hydrogen (˜15 psi, H₂-balloon). The crude material after filtrationmay be used directly and without further isolation in the next step ormay be purified by preparative HPLC to afford the title compound (21c).

Step D: tert-Butyl3-(5-amino-2-methoxy-phenyl)-4-(tert-butoxycarbonylamino)butanoate (21d)

Following the General Procedure of Description 15, tert-butyl3-(5-amino-2-methoxy-phenyl)-4-(tert-butoxycarbonylamino)butanoate (21d)is prepared from tert-butyl4-amino-3-(5-amino-2-methoxy-phenyl)butanoate (21c) (2.80 g, 10.0 mmol)in anhydrous dichloromethane (DCM) (30 mL) at about 0° C. (ice bath)with di-tert-butyl dicarbonate (Boc₂O) (2.29 g, 10.5 mmol) in thepresence of triethylamine (Et₃N, TEA) (1.67 mL, 1.21 g, 12.0 mmol) and acatalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP) (61 mg, 0.5mmol). Aqueous work-up and purification by silica gel columnchromatography with mixtures of ethyl acetate (EtOAc) and hexane yieldthe title compound (21d).

Step E: tert-Butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21e)

Variant A:

Following the General Procedure of Description 18, tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21e) is prepared from tert-butyl3-(5-amino-2-methoxy-phenyl)-4-(tert-butoxycarbonylamino)butanoate (21d)(3.81 g, 10.0 mmol) through reaction with ethylene oxide (12.5 mL, 11.0g, 100.0 mmol) in 15 mL of 50 vol.-% aqueous acetic acid (HOAc) for 24hours at room temperature to yield the title compound (21e) afteraqueous work-up and purification by silica gel chromatography.

Step F: tert-Butyl3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21f)

Following the General Procedure of Description 19 (Variant A),tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21f) is prepared from tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21e) (2.43 g, 5.0 mmol) through reaction with thionyl chloride (SOCl₂)(3.63 mL, 5.93 g, 50 mmol) in 25 mL of anhydrous chloroform (CHCl₃) for2 hours at reflux temperature to yield the title compound (21f) afterwork-up and purification by silica gel column chromatography using ethylacetate (EtOAc) and hexane mixtures.

Following the General Procedure of Description 19 (Variant B),tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21f) is prepared from tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21e) (2.43 g, 5.0 mmol) through reaction with phosphoryl chloride(POCl₃) (2.34 mL, 3.83 g, 25.0 mmol) in anhydrous benzene (20 mL) forabout 5 h at a temperature of about 80° C. to yield compound (21f) afterwork-up and purification by silica gel column chromatography using ethylacetate (EtOAc), and hexanes mixtures.

Following the General Procedure of Description 19 (Variant C),tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21f) is prepared from tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21e) (2.43 g, 5.0 mmol) through reaction with methanesulfonyl chloride(MsCl) (1.94 mL, 2.86 g, 25.0 mmol) in anhydrous pyridine (20 mL) for 2hours at 90° C. to yield the target compound (21f) after work-up andpurification by silica gel column chromatography using ethyl acetate(EtOAc) and hexane mixtures.

Following the General Procedure of Description 19 (Variant D),tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21f) is prepared from tert-butyl3-[5-(bis(2-hydroxyethyl)amino)-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21e) (2.43 g, 5.0 mmol) through reaction with triphenylphosphine (Ph₃P)(2.62 g, 10.0 mmol) and carbon tetrachloride (CCl₄) (1.45 mL, 2.31 g,15.0 mmol) in anhydrous dichloromethane (DCM) (20 mL) at roomtemperature for 8 hours to yield the target compound (21f) after work-upand purification by silica gel column chromatography using ethyl acetate(EtOAc) and hexane mixtures.

Step G: 4-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]butanoicacid (21)

Following the General Procedure of Description 23 (Variant B),4-amino-3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]butanoic acid(21) is prepared from tert-butyl3-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-4-(tert-butoxycarbonylamino)butanoate(21f) (505 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2 N HCl in Et₂O)(10 mL, 20 mmol) to yield the target compound (21) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 22 4-Amino-3-[5-[bis(2-chloroethyl)aminomethyl]-2-methyl-phenyl]butanoic acid (22)

Step A: Methyl (E)-3-(5-cyano-2-methyl-phenyl)prop-2-enoate (22a)

Following the General Procedure of Description 2 (Variant A), methyl(E)-3-(5-cyano-2-methyl-phenyl)prop-2-enoate (22a) is prepared fromcommercial 3-formyl-4-methyl-benzonitrile (2.90 g, 20.0 mmol), trimethylphosphonoacetate (4.04 mL, 4.55 g, 25.0 mmol), and anhydrous lithiumbromide (LiBr) (2.61 g, 30.0 mmol) in a mixture of triethylamine (Et₃N,TEA) (3.83 mL, 2.78 g, 27.5 mmol) and acetonitrile (MeCN) (20 mL).Purification by silica gel column chromatography with a mixture of ethylacetate (EtOAc) and hexane furnish the title compound (22a).

Step B: Methyl 3-(5-cyano-2-methyl-phenyl)-4-nitro-butanoate (22b)

Following the General Procedure Description 3, methyl3-(5-cyano-2-methyl-phenyl)-4-nitro-butanoate (22b) was prepared frommethyl (E)-3-(5-cyano-2-methyl-phenyl)prop-2-enoate (22a) (3.09 g, 15.0mmol) in a mixture of nitromethane (MeNO₂) (8.2 mL, 9.34 g, 153 mmol)and acetonitrile (MeCN) (15 mL) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.29 mL, 2.33 g, 15.3 mmol).Purification by silica gel column chromatography with mixtures of ethylacetate (EtOAc) and hexane furnishes the title compound (22b).

Step C: 4-[5-(Aminomethyl)-2-methyl-phenyl]pyrrolidin-2-one (22c)

Adapting a literature known protocol (Osby and Ganem, Tetrahedron Lett.,1985, 26(52), 6413-6416), a mixture of nickel(II) dichloride hexahydrate(NiCl₂.6H₂O) (1.19 g, 5.0 mmol) in methanol (MeOH) (80 mL) is sonicatedto effect complete dissolution. Solid sodium borohydride (NaBH₄) (567mg, 15.0 mmol) is added in small portions upon a black precipitate(Ni₂B) is generated immediately and hydrogen gas is generated(exotherm!). After about 30 min, a solution of methyl3-(5-cyano-2-methyl-phenyl)-4-nitro-butanoate (22b) (2.62 g, 10.0 mmol)in MeOH (20 mL) is added followed by portion-wise addition of more NaBH₄(1.13 g, 30.0 mmol) at room temperature. The reaction is monitored byTLC and/or LCMS till completion. The reaction mixture is filtered overCelite® 545 and the filter residue is washed with additional MeOH. Thecombined filtrates are partially evaporated under reduced pressure usinga rotary evaporator and the crude material is heated to 50-60° C. tocomplete the lactamization. Purification by silica gel columnchromatography with mixtures of (MeOH) and dichloromethane (DCM)furnishes the title compound4-[5-(aminomethyl)-2-methyl-phenyl]pyrrolidin-2-one (22c).

Step D: 4-[5-[Bis(2-chloroethyl)aminomethyl]-2-methyl-phenyl]pyrrolidin-2-one (22d)

Following the General Procedure of Description 17 (Variant A),4-[5-[bis(2-chloroethyl)aminomethyl]-2-methyl-phenyl]pyrrolidin-2-one(22d) is prepared from4-[5-(aminomethyl)-2-methyl-phenyl]pyrrolidin-2-one (22c) (1.02 g, 5.0mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (3.82 mL, 30.0mmol), and sodium cyanoborohydride (NaBH₃CN) (1.57 g, 25.0 mmol) in amixture of methanol (MeOH) (20 mL) and trifluoroacetic acid (TFA) (10mL). Purification by silica gel column chromatography with ethyl acetate(EtOAc) furnishes the title compound (22d).

Step E:4-Amino-3-[5-[bis(2-chloroethyl)aminomethyl]-2-methyl-phenyl]butanoicacid (22)

Following the General Procedure of Description 22,4-amino-3-[5-[bis(2-chloroethyl)aminomethyl]-2-methyl-phenyl]butanoicacid (22) is prepared from4-[5-[bis(2-chloroethyl)aminomethyl]-2-methyl-phenyl]pyrrolidin-2-one(22d) (659 mg, 2.0 mmol) by hydrolysis in a mixture of concentratedhydrochloric acid (HCl) (about 10 mL) and 1,4-dioxane at refluxtemperature for about 14 hours to furnish the title compound (22) as adihydrochloride salt after isolation using evaporation andlyophilization. The material is purified by preparative RP-HPLC using awater/acetonitrile/0.1 vol-% formic acid gradient to afford the titlecompound (22) as a dihydrochloride salt after final lyophilization ofthe solvents in the presence of an excess of 1.0 M hydrochloric acid(HCl). Various batches of mono- or dihydrochloride salts of (22) can beprepared by primary lyophilization of solutions of (22) in aqueousacetonitrile (MeCN) containing either 1.0 eq. of 1.0 N hydrochloric acid(HCl) or an excess of 1.0 N or higher concentrated hydrochloric acid(HCl).

Example 234-Amino-3-[5-[bis(2-chloroethyl)carbamoyloxy]-2-methyl-phenyl]butanoicacid (23)

Step A: tert-Butyl (E)-3-(5-benzyloxy-2-methyl-phenyl)prop-2-enoate(23a)

Following the General Procedure of Description 2 (Variant A), tert-butyl(E)-3-(5-benzyloxy-2-methyl-phenyl)prop-2-enoate (23a) is prepared5-benzyloxy-2-methyl-benzaldehyde (prepared from commercial methyl5-hydroxy-2-methyl-benzoate in 4 steps (i) BnBr, DMF, K₂CO₃; ii) aq.LiOH, MeOH/THF; iii) BH₃.SMe₂, THF, Δ; iv) MnO₂, CH₂Cl₂) using methodswell known in the art or described herein) (4.52 g, 20.0 mmol),commercial tert-butyl diethyl phosphonoacetate (5.87 mL, 6.31 g, 25.0mmol), and anhydrous lithium bromide (LiBr) (2.61 g, 30.0 mmol) in amixture of triethylamine (Et₃N, TEA) (3.83 mL, 2.78 g, 27.5 mmol) andacetonitrile (MeCN) (20 mL). Purification by silica gel columnchromatography with a mixture of ethyl acetate (EtOAc) and hexanefurnishes the title compound (23a).

Step B: tert-Butyl 3-(5-benzyloxy-2-methyl-phenyl)-4-nitro-butanoate(23b)

Following the General Procedure of Description 3, tert-butyl3-(5-benzyloxy-2-methyl-phenyl)-4-nitro-butanoate (23b) is prepared fromtert-butyl (E)-3-(5-benzyloxy-2-methyl-phenyl)prop-2-enoate (23a) (4.87g, 15.0 mmol) in a mixture of nitromethane (MeNO₂) (10 mL, 11.4 g, 187mmol) and acetonitrile (MeCN) (15 mL) in the presence of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.24 mL, 2.28 g, 15.3 mmol).Aqueous work-up and purification by silica gel column chromatographywith mixtures of ethyl acetate (EtOAc) and hexane afford the titlecompound (23b).

Step C: tert-Butyl3-(5-benzyloxy-2-methyl-phenyl)-4-(tert-butoxycarbonyl-amino)butanoate(23c)

Following the General Procedure of Description 12, tert-butyl3-(5-benzyloxy-2-methyl-phenyl)-4-(tert-butoxycarbonyl-amino)butanoate(23c) was prepared by reduction of tert-butyl3-(5-benzyloxy-2-methyl-phenyl)-4-nitro-butanoate (23b) (5.78 g, 15.0mmol) with nickel acetate tetrahydrate (Ni(OAc)₂.4H₂O) (747 mg, 3.0mmol) and sodium borohydride (NaBH₄) (2.27 g, 60 mmol) in a mixture ofacetonitrile (MeCN) (45 mL) and water (4.5 mL) containing di-tert-butyldicarbonate (Boc₂O) (3.49 g, 16.0 mmol) and catalytic amount of4-(N,N-dimethylamino)pyridine (DMAP) (183 mg, 1.5 mmol, 10 mol-%).Purification by silica gel column chromatography using a mixture ofethyl acetate (EtOAc) and hexane afford the target compound (23c).

Step D: tert-Butyl4-(tert-butoxycarbonylamino)-3-(5-hydroxy-2-methyl-phenyl)butanoate(23d)

Following the General Procedure of Description 16, tert-butyl4-(tert-butoxycarbonylamino)-3-(5-hydroxy-2-methyl-phenyl)butanoate(23d) is prepared by reduction of tert-butyl3-(5-benzyloxy-2-methyl-phenyl)-4-(tert-butoxycarbonyl-amino)butanoate(23c) (3.85 g, 10.0 mmol) under an atmosphere of hydrogen (˜15 psi,H₂-balloon) and in the presence of 10 wt-% palladium on charcoal (Pd/C)containing ˜50 wt-% water (˜1.5 g) in methanol (MeOH) (40 mL). Afterfiltration over Celite® 545, evaporation of solvents and aqueouswork-up, the crude material may be used directly in the next step or ispurified by silica gel column chromatography with mixtures of ethylacetate (EtOAc) and hexane afford the title compound (23d).

Step E: tert-Butyl3-[5-[bis(2-chloroethyl)carbamoyloxy]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(23e)

Adapting a literature known protocol (U.S. Pat. No. 3,299,104),tert-butyl3-[5-[bis(2-chloroethyl)carbamoyloxy]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(23e) is prepared through carbamoylation of tert-butyl4-(tert-butoxycarbonylamino)-3-(5-hydroxy-2-methyl-phenyl)butanoate(23d) (731 mg, 2.0 mmol) with commercial N,N-bis(2-chloroethyl)carbamoylchloride (439 μL, 614 mg, 3.0 mmol) in anhydrous pyridine (15 mL) atabout 0° C. The reaction mixture is stirred with gradual warming to roomtemperature. The reaction is monitored by TLC and/or LC/MS tocompletion. Excess of the carbamoyl chloride is destroyed with crushedice. Aqueous work-up followed by purification through silica gel columnchromatography afford the title compound.

Step F:4-Amino-3-[5-[bis(2-chloroethyl)carbamoyloxy]-2-methyl-phenyl]butanoicacid (23)

Following the General Procedure of Description 22,4-amino-3-[5-[bis(2-chloroethyl)carbamoyloxy]-2-methyl-phenyl]butanoicacid (23) is prepared from tert-butyl3-[5-[bis(2-chloroethyl)carbamoyloxy]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)butanoate(23e) (533 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2 N HCl in Et₂O)(10 mL, 20 mmol) to yield the target compound (23) as a solidhydrochloride salt after evaporation of the solvents and lyophilizationfrom an aqueous solution. The material may be further purified bypreparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 244-Amino-3-[4-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]-3-methyl-butanoicacid (24)

Step A: tert-Butyl4-(tert-butoxycarbonylamino)-3-[4-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]-3-methyl-butanoate(24a)

Adapting literature known protocols (Tercel, et al., J. Med. Chem. 1995,38, 1247-1252; U.S. Pat. No. 5,602,278; Kirkpatrick, et al., Anti-CancerDrugs, 1994, 5, 467-472; and U.S. Pat. No. 7,399,785), tert-butyl4-(tert-butoxycarbonylamino)-3-[4-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]-3-methyl-butanoate(24a) is prepared by adding 3-chloroperoxybenzoic acid (1.42 g, 80 wt-%,6.6 mmol) to a solution of tert-butyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7o) (2.52 g, 5.0 mmol) in dichloromethane (DCM) (30 mL) at about roomtemperature for about 2 h. The reaction is followed by TLC and/or LC/MStill completion. After quenching with a saturated aqueous solution ofsodium hydrogencarbonate (NaHCO₃), the reaction mixture is extractedwith DCM (3×). Further aqueous work-up and purification by silica gelcolumn chromatography yield the title compound (24a).

Step B:4-Amino-3-[4-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]-3-methyl-butanoicacid (24)

Following the General Procedure of Description 22,4-amino-3-[4-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]-3-methyl-butanoicacid (24) is prepared from tert-butyl4-(tert-butoxycarbonylamino)-3-[4-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]-3-methyl-butanoate(24a) (520 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2.0 N HCl in Et₂O)(10 mL, 20 mmol) to yield the target compound (24) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 254-[1-(Aminomethyl)-3-hydroxy-1-methyl-3-oxo-propyl]-N,N-bis(2-chloroethyl)-3-methyl-benzeneamineoxide (25)

Step A:4-[3-tert-Butoxy-1-[(tert-butoxycarbonylamino)methyl]-1-methyl-3-oxo-propyl]-N,N-bis(2-chloroethyl)-3-methyl-benzeneamineoxide (25a)

Adapting literature known protocols (Tercel, et al., J. Med. Chem. 1995,38, 1247-1252; and U.S. Pat. No. 7,399,785), peracetic acid (H₃CCO₃H) isfreshly prepared by adding hydrogen peroxide (H₂O₂) (1.5 mL of a 35 wt-%aqueous solution, 14.0 mmol) dropwise to acetic anhydride (Ac₂O) (1.52mL, 1.65 g, 16.0 mmol). When the reaction mixture is homogeneous, asolution of tert-butyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-4-(tert-butoxycarbonylamino)-3-methyl-butanoate(7o) (1.66 g, 3.29 mmol) in dichloromethane (DCM) (20 mL) is added withvigorous stirring at about room temperature for about 2 h. The reactionis followed by TLC and/or LC/MS to completion. The reaction is quenchedwith 2.0 N hydrochloric acid (HCl), and the aqueous layer separated andrepeatedly washed with DCM until the organic extracts are colorless. Theaqueous phase is evaporated to dryness under reduced pressure, driedover anhydrous sodium sulfate (Na₂SO₄), and partially reduced in volume.Diethyl ether (Et₂O) is added to separate the title compound4-[3-tert-butoxy-1-[(tert-butoxycarbonylamino)methyl]-1-methyl-3-oxo-propyl]-N,N-bis(2-chloroethyl)-3-methyl-benzeneamineoxide (25a). The material may be purified by silica gel columnchromatography using ethyl acetate (EtOAc), methanol (MeOH), and hexanemixtures.

Step B:4-[1-(Aminomethyl)-3-hydroxy-1-methyl-3-oxo-propyl]-N,N-bis(2-chloroethyl)-3-methyl-benzeneamineoxide (25)

Following the General Procedure of Description 22,4-[1-(aminomethyl)-3-hydroxy-1-methyl-3-oxo-propyl]-N,N-bis(2-chloroethyl)-3-methyl-benzeneamineoxide (25) is prepared from4-[3-tert-butoxy-1-[(tert-butoxycarbonylamino)methyl]-1-methyl-3-oxo-propyl]-N,N-bis(2-chloroethyl)-3-methyl-benzeneamineoxide (25a) (520 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2 N HCl inEt₂O) (10 mL, 20 mmol) to yield the target compound (25) as an soliddihydrochloride salt (25.2HCl) after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 26 LAT1 Uptake Inhibition Assays

The ability of compounds to interact with LAT1 was measured using aradiolabeled competition uptake assay with [³H]-Gabapentin (GP) in96-well plates with LLCPK cells conditionally expressing hLAT1. 5×10⁴cells/well were plated in white, clear bottom plates in the presence orabsence of tetracycline or doxcycline to induce hLAT1 expression. Thenext day, cells were treated with butyrate to stimulate additional hLAT1expression. On the third day, the cells were washed and then incubatedwith 50,000 cpm of [³H]-GP in PBS in the presence or absence of 1 mM oftest compound in at least triplicate for 15 min. At end of the assaytime, the incubation solution was removed, and the plates were washedthree times with 100 μL of ice-cold PBS. 150 μL of scintillation fluidwas added to each well, and the radioactivity retained within the cellswas measured on a 96-well scintillation counter. The data are expressedas a percent of specific [³H]-GP uptake. Unlabeled GP and other largeamino acids (phenylalanine and leucine) were used as controls.

The ability of various compounds to interact with LAT1 was assessed bymeasuring the inhibition of [3H]-GP uptake into LAT1-expressing cells inthe presence of 1 mM test compound. Unlabeled GP and phenylalanine (Phe)and leucine (Leu) were used as controls. After incubation for 15 min,cells were washed, scintillation fluid added, and cell-boundradioactivity determined in a scintillation counter. Data are expressedas a percent of specific GP uptake.

The specific uptake of radiolabeled gabapentin into LAT1-expressingcells was inhibited by 1 mM of unlabeled gabapentin, phenylalanine,leucine, and the compounds of Examples 1-7 and 26. Treatment withgabapentin, phenylalanine, leucine, and the compounds of Example 5 andExample 7 resulted in specific uptake of less than 10%. The compounds ofExamples 1-4, 6, and 26 resulted in specific uptake of greater than 20%but less than 65% at this concentration. The specific uptake ofradiolabeled gabapentin in the absence of any compound was 100%.

Example 27 LAT1-Specific In Vitro Cytotoxicity Assays

The LAT1-specific in vitro cytotoxicity of compounds was assessed byusing a modified clonigenic assay in 96-well plates with LLCPK cellsconditionally expressing hLAT1. One-thousand (1000) cells/well wereplated in clear bottom plates in the presence or absence of tetracyclineor doxcycline to induce hLAT1 expression. The next day, cells weretreated with butyrate to stimulate additional hLAT1 expression. On thethird day, cells were washed and incubated with various concentrationsof test compounds in PBS in at least quadruplicate for 30 minutes. Atthe end of the treatment, test compounds were removed and growth mediawas added to the cells. Clonal populations were allowed to grow untilthe control wells (mock treatment) were near confluency (7 to 10 days).Cell growth was quantified by fixing and staining the cells post-washwith crystal violent in glutaraldehye, washing away unadhered dye,solubilizing the stained cells in acetic acid and monitoring absorbanceat 530 nm. Data from each test concentration were expressed as thepercent of live, mock-treated controls (% surviving cells). LAT1specificity was determined by the differential toxicity in cells induced(LAT1+) vs. non-induced (no LAT1) to express hLAT1. Melphalan, aN-mustard compound, was used as a control.

The LAT1-specific cytotoxicity of various compounds was assessed bytreating cells expressing or not expressing LAT1 with 3 μM of testcompound for 30 min. Melphalan was used as a control compound. Followingtreatment, cells were washed and growth media was added. Surviving cellswere allowed to proliferate for 7-10 days, and then stained andquantified.

The percent surviving cells for melphalan and the compound of Example 6was about the same in cells expressing LAT1 and in cells not expressingLAT1. The percent surviving cells for the compounds of Examples 2-5 and7 was significantly reduced by at least 20% in cells expressing LAT1compared to cells not expressing LAT1.

Example 28 In Vivo Tumor Growth Suppression Assays

The ability to suppress the growth of tumors in vivo was measured usinga B16 efficacy model (Kato, et al., Cancer Res., 1994, 54, 5143-5147).Briefly, the hind flank of C57BL/6 mice were injected with 5×10⁵ B16melanoma cells subcutaneously. Once the tumors reached 40 mm³, animalswere separated into various treatment arms (n=5) and dosed IP daily withvehicle or test compound (5 mg/kg and 10 mg/kg) for 12 days. Tumor sizeswere monitored every third day for up to three weeks. Melphalan (2.5mg/kg) was used as a control compound. The results are presented inTable 1.

TABLE 1 Tumor Suppression by QBS Compounds in vivo. Tumor Growth (%Control) End of 5 days Treatment dosing post-dosing Vehicle 100 100Example 5 13 14 Example 7 38 50 Melphalan 33 56

Finally it should be noted that there are alternative ways ofimplementing the embodiments disclosed herein. Accordingly, the presentembodiments are to be considered as illustrative and not restrictive,and the claims are not to be limited to the details given herein, butmay be modified within the scope and equivalents thereof.

What is claimed is:
 1. A compound of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein: R¹ comprises anitrogen mustard; R⁴ is selected from hydrogen, deuterio, C₁₋₆ alkyl,and C₁₋₆ alkoxy; each of the other of R², R³, and R⁵ is independentlyselected from hydrogen, deuterio, and C₁₋₆ alkyl; R⁶ is —COOH; each R⁷is independently selected from hydrogen and deuterio; R⁸ is selectedfrom hydrogen, deuterio, and C₁₋₆ alkyl; and L is selected from a bondand —CH₂—.
 2. The compound of claim 1, wherein, R⁴ is C₁₋₆ alkyl; eachof R², R⁴, and R⁵ is hydrogen; and R⁸ is methyl.
 3. The compound ofclaim 1, wherein, R⁴ is methyl; each of R², R³, and R⁵ is hydrogen; andR⁸ is methyl.
 4. The compound of claim 1, wherein, the nitrogen mustardis a moiety of Formula (2):

wherein, A is selected from a bond (“-”), oxygen (—O—), sulfur (—S—),amino (—NR¹⁰—), methylene (—CH₂—), methyleneoxy (—CH₂—O—), oxycarbonyl(—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl (—NR¹⁰—C(═O)—),oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl (—S—C(═S)—),aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl (—CH₂—O—C(═O)—),methylenethiocarbonyl (—CH₂—S—C(═O)—), methyleneaminocarbonyl(—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl (—CH₂—O—C(═S)—),methylenethiothiocarbonyl (—CH₂—S—C(═S)—), methyleneaminothiocarbonyl(—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—), methylencarbonyl (—CH₂—C(═O)—),thiocarbonyl (—C(═S)—), and methylenethiocarbonyl (—CH₂—C(═S)—); Z isselected from a bond (“-”) and oxygen (—O—); Q is selected from —O⁻ (anegatively charged oxygen atom) that is bound to a positively chargednitrogen atom) and a free electron pair (:), with the proviso that whenQ is —O⁻ (a negatively charged oxygen atom that is bound to a positivelycharged nitrogen atom), then A is selected from a bond (“-”) andmethylene (—CH₂—), Z is a bond (“-”), and N is positively charged; eachR¹¹ is independently selected from hydrogen, deuterio, and C₁₋₃ alkyl;and each R⁹ is independently selected from fluoro (—F), chloro (—Cl),bromo (—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from C₁₋₄ alkyl), C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰,wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl), and (substituted)aryl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₆₋₁₀ aryl). 5.The compound of claim 1, wherein the nitrogen mustard is a moiety havingthe structure of formula (2a):-A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)  (2a) wherein, A isselected from a bond (“-”), methylene (—CH₂—), oxygen (—O—),methyleneoxy (—CH₂—O—), carbonyl (—C(═O)—), methylenecarbonyl(—CH₂—C(═O)—), oxycarbonyl (—O—C(═O)—), and methyleneoxycarbonyl(—CH₂—O—C(═O)—); Z is selected from a bond (“-”) and oxygen (—O—); Q isselected from —O⁻ (a negatively charged oxygen atom that is bound to apositively charged nitrogen atom) and a free electron pair (:); each R¹¹is independently selected from hydrogen and deuterio; and each R⁹ isindependently selected from fluoro (—F), chloro (—Cl), bromo (—Br), iodo(—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is C₁₋₄ alkyl),C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isC₁₋₄(per)fluoroalkyl), and (substituted) aryl sulfonate (—OSO₂R⁴⁰,wherein R⁴⁰ is C₆₋₁₀ aryl); each of the other of R¹, R², R³, R⁴, and R⁵is independently selected from hydrogen, deuterio, halogen, —OH,—N(R¹⁰)₂, —NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, C₁₋₄ alkylsulfanyl, C₁₋₄alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₆ alkyl, substituted C₁₋₆ alkyl,C₃₋₆ cycloalkyl, and substituted C₃₋₆ cycloalkyl.
 6. The compound ofclaim 1, wherein the nitrogen mustard is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein, each R⁹is independently selected chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).7. The compound of claim 1, wherein the nitrogen mustard has thestructure —N—(—CH₂—CH₂—Cl)₂.
 8. The compound of claim 1, which exhibits:a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L); and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 50% that of L-leucine measured at an extracellularconcentration of 1 mM (1 mmol/L).
 9. A pharmaceutical compositioncomprising the compound of claim 1 and a pharmaceutically acceptablevehicle.